Inbred miniature swine and uses thereof

ABSTRACT

The invention provides a swine which is homozygous for a major histocompatibility complex haplotype and at least 60% homozygous at all other genetic loci and such animal is propagatable, and a cell or an organ derived therefrom. The invention also provides a method for providing a swine which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other genetic loci are homozygous, as well as a method of inducing tolerance in a recipient mammal of a first species to a graft from a donor mammal of a second species.

RELATED APPLICATIONS

This application is a divisional application of and claims priority fromU.S. patent application Ser. No. 10/224,294, filed on Aug. 20, 2002,which is a continuation of U.S. patent application Ser. No. 09/378,684,filed on Aug. 20, 1999, now U.S. Pat. No. 6,469,229, which claimsbenefit from U.S. Provisional Patent Application Ser. No. 60/097,423,filed on Aug. 20, 1998. The contents of these prior applications arehereby incorporated by reference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

The major histocompatibility complex (MHC) is a set of linked geneswhich code for cell surface proteins involved in transplant rejection.The MHC contains three types of genes, class I, II and III (Klein J. etal.: Immunology: The Science of Self-Nonself Discrimination, pp. 687,1984, John Wiley, Somerset, N.J.).

In humans, class I genes encode polymorphic 44,000 dalton glycoproteinchains that associate with a nonpolymorphic 12,000 dalton light chain,β2-microglobulin, and which are expressed on most cells of the body.Typical class I MHC genes are involved in regulating immune to viralinfections (Zinkemagal R. M. et al. (1979) Adv. Immunol. 27:52-72).

In humans, the class II MHC antigens are cell surface glycoproteinscomposed of an α chain of approximately 35,000 daltons and a β chain ofabout 28,000 that are expressed only on subsets of immunologicallyactive cells, such as β lymphocytes and macrophages.

Class III MHC genes code for serum proteins such as complement (C′).

The MHC loci in swine are known as the swine leukocyte antigens (SLA).In 1970, Vaiman et al., (Vaiman M. et al. (1970) Transplantation 10:155-161) and Viza et al. (Viza D. et al. (1970) Nature 227:949-951)provided descriptions of the SLA complex. These groups developed panelsof SLA typing reagents (Vaiman M. et al. (1979) Immunogenetics9:353-361) by preparing antisera of defined specificity as well as bycharacterizing cells of known SLA type (homozygous typing cells) for usein mixed lymphocyte complex, to chromosome 7 (Geffrotin C. et al. (1984)Ann Genet (Parix) 27:213-219). The class I swine MHC loci are designatedSLA-A,B,C. The class II swine MHC loci are designated SLA-DR, DQ.Because there are numerous genes coded by the SLA complex and becauseusually they are inherited as a unit, haplotype designations have beendeveloped. For example, the SLAa haplotype codes for SLA-A^(a)B^(a)C^(a)DR^(a)DQ^(a) alleles.

Miniature swine are a good model for organ transplantation studiesbecause of their breeding characteristics which make them one of fewlarge animals in which genetics can be manipulated in a reasonable time,and also because of their size which permits surgical manipulationssimilar to those humans.

SUMMARY OF THE INVENTION

The invention provides a genetically defined, large animal, useful,e.g., as an organ, tissue, or cell, donor, which is homozygous at swineleukocyte antigens (SLA) A, B, C, DR, and DQ, and preferably in which asufficient number of all other genetic loci are homozygous such that anorgan, tissue, or cell, from one animal can be used to prolongacceptance in a recipient, e.g., a xenorecipient, of an organ, tissue,or cell, from a second animal from a herd of such animals, or such thatprolongation of acceptance (e.g., by the induction of tolerance) in arecipient, e.g., a xenorecipient, of an organ, tissue, or cell, from oneanimal of the herd also provides prolongation of acceptance of an organ,tissue, or cell, from a second animal of the herd.

Accordingly, the invention features, a swine, preferably a miniatureswine, which is homozygous at swine leukocyte antigens (SLA) A, B, C,DR, and DQ, and in which at least 60% of all other genetic loci arehomozygous. In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%,90%, 95% or more, of all other genetic loci in the swine are homozygous.

In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C,DR, and DQ can be of haplotype a (A^(a), B^(a), C^(a), DR^(a), DQ^(a)),haplotype c (A^(c), B^(c), C^(c), DR^(c), DQ^(c)) haplotype d (A^(d),B^(d), C^(d), DR^(d), DQ^(d)), haplotype g (A^(g), B^(g), C^(g), DR^(g),DQ^(g)), haplotype h (A^(h), B^(h), C^(h), DR^(h), DQ^(h)), or haplotypej (A^(j), B^(j), C^(j), DR^(j), D^(j)).

In preferred embodiments, the swine is capable of reproduction, i.e.,the animal can produce functional gametes.

In another aspect, the invention features, a cell or a preparation ofsuch cells, from a swine, preferably a miniature swine, which ishomozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and inwhich at least 60% of all other genetic loci are homozygous.

In preferred embodiments, the swine cell is an embryonic stem cell. Inother preferred embodiments, the swine cell can be a hematopoietic stemcell, e.g., a cord blood hematopoietic stem cell, a bone marrowhematopoietic stem cell, or a fetal or neonatal liver or spleenhematopoietic stem cell; a differentiated blood cell, e.g., a myeloidcell, a megakaryocyte, a monocyte, a granulocyte, an eosinophil, anerythroid cell, a lymphoid cell, such as a B lymphocyte or a Tlymphocyte; a pluripotent hematopoietic stem cell, e.g., a hematopoieticprecursor, a burst-forming units-erythroid (BFU-E), a colony formingunit-erythroid (CFU-E), a colony forming unit-megakaryocyte (CFU-Meg), acolony forming unit-granulocyte-monocyte (CFU-GM), a colony formingunit-eosinophil (CFU-Eo), or a colony formingunit-granulocyte-erythrocyte-megakaryocyte-monocyte (CFU-GEMM); a swinecell other than a hematopoietic stem cell or other blood cell; a swinethymic cell, e.g., a swine thymic stromal cell; a bone marrow stromalcell; a swine liver cell; a swine kidney cell; a swine epithelial cell;a swine muscle cell, e.g., a heart cell; or a dendritic cell orprecursor thereof.

In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore, of all other genetic loci in the swine cell are homozygous.

In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C,DR, and DQ can be of haplotype a (A^(a), B^(a), C^(a), DR^(a), DQ^(a)),haplotype c (A^(c), B^(c), C^(c), DR^(c), DQ^(c)), haplotype d (A^(d),B^(d), C^(d), DR^(d), DQ^(d)), haplotype g (A^(g), B^(g), C^(g), DR^(g),DQ^(g)), haplotype h (A^(h), B^(h), C^(h), DR^(h), DQ^(h)), or haplotypej (A^(j), B^(j), C^(j), DR^(j), DQ^(j)′).

In another aspect, the invention features, an isolated cell nucleus froma swine cell, preferably a miniature swine cell, which is homozygous atswine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which atleast 60% of all other genetic loci are homozygous. In preferredembodiments, the cell nucleus is from an undifferentiated cell. In otherembodiments, the cell nucleus is from a differentiated cell.

In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore, of all other genetic loci in the swine cell nucleus arehomozygous.

In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C,DR, and DQ can be of haplotype a (A^(a), B^(a), C^(a), DR^(a), DQ^(a)),haplotype c (A^(c), B^(c), C^(c), DR^(c), DQ^(c)), haplotype d (A^(d),B^(d), C^(d), DR^(d), DQ^(d)), haplotype g (A^(g), B^(g), C^(g), DR^(g),DQ^(g)), haplotype h (A^(h), B^(h), C^(h), DR^(h), DQ^(h)), or haplotypej (A^(j), B^(j), C^(j), DR^(j), DQ^(j)).

In another aspect, the invention features, an isolated organ, or atissue, from a swine, preferably a miniature swine, which swine ishomozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and inwhich at least 60% of all other genetic loci are homozygous.

In preferred embodiments, the organ can be an organ of thegastrointestinal tract, a liver, a kidney, a pancreas, a stomach, aspleen, or a gallbladder; a sensory organ, e.g., an eye; a lung; onorgan or tissue of the circulatory system, e.g., a heart. In otherpreferred embodiments, the tissue can be connective tissue; epithelialtissue, e.g., skin; muscle tissue; osseous tissue; vascular tissue,e.g., a blood vessel; or occular tissue, e.g., lens tissue.

In preferred embodiments, the isolated organ or tissue is from apostnatal animal, e.g., a juvenile or adult animal, or a prenatalanimal, e.g., a fetus or an embryo.

In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore, of all other genetic loci in the swine are homozygous.

In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C,DR, and DQ can be of haplotype a (A^(a), B^(a), C^(a), DR^(a), DQ^(a)),haplotype c (A^(c), B^(c), C^(c), DR^(c), DQ^(c)), haplotype d (A^(d),B^(d), C^(d), DR^(d), DQ^(d)), haplotype g (A^(g), B^(g), C^(g), DR^(g),DQ^(g)), haplotype h (A^(h), B^(h), C^(h), DR^(h), DQ^(h)), or haplotypej (A^(j), B^(j), C^(j), DR^(j), DQ^(j)).

In another aspect, the invention features, a hematopoietic stem cellpreparation, e.g., a bone marrow stem cell preparation, from a swine,preferably a miniature swine, which swine is homozygous at swineleukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60%of all other genetic loci are homozygous.

In preferred embodiments, the hematopoietic stem cell preparation isfrom a postnatal animal, e.g., a juvenile or adult animal, or a prenatalanimal, e.g., a fetus or an embryo.

In preferred embodiments, the preparation includes hematopoietic stemcells from cord blood, the liver, or spleen.

In preferred embodiments, the preparation is a bone marrow preparationwhich includes immature bone marrow cells, e.g., undifferentiatedhematopoietic stem cells, in addition to other bone marrow components.In other preferred embodiments, the bone marrow preparation is composedof isolated undifferentiated hematopoietic stem cells.

In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore, of all other genetic loci in the swine are homozygous.

In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C,DR, and DQ can be of haplotype a (A^(a), B^(a), C^(a), DR^(a), DQ^(a)),haplotype c (A^(c), B^(c), C^(c), DR^(c), DQ^(c)) haplotype d (A^(d),B^(d), C^(d), DR^(d), DQ^(d)), haplotype g (A^(g), B^(g), C^(g), DR^(g),DQ^(g)), haplotype h (A^(h), B^(h), C^(h), DR^(h), DQ^(h)), or haplotypej (A^(j), B^(j), C^(j), DR^(j), DQ^(j)).

In another aspect, the invention features, a herd of swine, preferablyminiature swine, in which the animals are homozygous at swine leukocyteantigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of allother genetic loci are homozygous. In preferred embodiments, the herd ofswine includes at least one male swine and at least one female swinecapable of reproduction, e.g., at least one male and one female whichcan produce functional gametes.

In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore, of all other genetic loci in the swine in the herd are homozygous.

In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C,DR, and DQ can be of haplotype a (A^(a), B^(a), C^(a), DR^(a), DQ^(a)),haplotype c (A^(c), B^(c), C^(c), DR^(c), DQ^(c)) haplotype d (A^(d),B^(d), C^(d), DR^(d), DQ^(d)), haplotype g (A^(g), B^(g), C^(g), DR^(g),DQ^(g)), haplotype h (A^(h), B^(h), C^(h), DR^(h), DQ^(h)), or haplotypej (A^(j), B^(j), C^(j), DR^(j), DQ^(j)).

In another aspect, the invention features, a method for providing aswine, preferably a miniature swine, which is homozygous at swineleukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60%of all other genetic loci are homozygous. The method includes: providinga first swine which is homozygous at swine leukocyte antigens (SLA) A,B, C, DR, and DQ but which is preferably homozygous at less than 20%,30%, 50%, or 75% of all other loci;

(1) providing a second swine which is homozygous at swine leukocyteantigens (SLA) A, B, C, DR, and DQ, which is of the same haplotype asthe first swine, but which is preferably homozygous at less than 20%,30%, 50%, or 75% of all other loci, which is preferably not a sibling,parent or offspring of the first swine;

(2) mating the first and second swine to provide an offspring;

(3) mating the offspring to a swine which is homozygous at swineleukocyte antigens (SLA) A, B, C, DR, and DQ, which is of the samehaplotype as the first swine but which is preferably homozygous at lessthan 20%, 30%, 50%, or 75% of all other loci, which is preferably not asibling, parent or offspring of the offspring;

(4) repeating step (3) for at least 18 generations;

(5) performing a brother sister mating from the offspring of the finalmating of step (4) to produce at least on male and one female sibling;

(6) performing brother sister matings form the siblings of step (5) andfor at least 5 additional generations;

to thereby provide a swine which is homozygous at swine leukocyteantigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of allother genetic loci are homozygous.

In preferred embodiments, the swine, which is homozygous at swineleukocyte antigens (SLA) A, B, C, DR, and DQ, is mated in nonbrother-sister matings for at least 10, 15, 16, 17, 18, 19, 20, or 25generations, and then mated in brother-sister matings for at least 4, 5,6, 7, 8, 9, or 10 generations.

In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore, of all other genetic loci in the swine are homozygous.

In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C,DR, and DQ can be of haplotype a (A^(a), B^(a), C^(a), DR^(a), DQ^(a)),haplotype c (A^(c), B^(c), C^(c), DR^(c), DQ^(c)) haplotype d (A^(d),B^(d), C^(d), DR^(d), DQ^(d)), haplotype g (A^(g), B^(g), C^(g), DR^(g),DQ^(g)), haplotype h (A^(h), B^(h), C^(h), DR^(h), DQ^(h)), or haplotypej (A^(j), B^(j), C^(j), DR^(j), DQ^(j)).

In preferred embodiments, the swine is capable of reproduction, i.e.,the animal can produce functional gametes.

In another aspect, the invention features, a swine, preferably aminiature swine, made by a method described herein.

In another aspect, the invention features, a method of providing aswine, preferably a miniature swine, which is homozygous at swineleukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60%of all other genetic loci are homozygous. The method includes mating amale swine which is homozygous at swine leukocyte antigens (SLA) A, B,C, DR, and DQ, and in which at least 60%. of all other genetic loci arehomozygous, with a female swine which is homozygous at swine leukocyteantigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of allother genetic loci are homozygous, thereby providing a swine which ishomozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and inwhich at least 60% of all other genetic loci are homozygous.

In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore, of all other genetic loci of one or more of and more preferablyall of the swine, the male swine, and the female swine are homozygous.

In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C,DR, and DQ can be of haplotype a (A^(a), B^(a), C^(a), DR^(a), DQ^(a)),haplotype c (A^(c), B^(c), C^(c), DR^(c), DQ^(c)), haplotype d (A^(d),B^(d), C^(d), DR^(d), DQ^(d)), haplotype g (A^(g), B^(g), C^(g), DR^(g),DQ^(g)), haplotype h (A^(h), B^(h), C^(h), DR^(h), DQ^(h)), or haplotypej (A^(j), B^(j), C^(j), DR^(j), DQ^(j)). In particularly preferredembodiments the halpotype of swine, the male swine, and the female swineare the same.

In preferred embodiments, the swine is capable of reproduction, i.e.,the animal can produce functional gametes.

In another aspect, the invention features, a method of providing aswine, preferably a miniature swine, which is homozygous at swineleukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60%of all other genetic loci are homozygous.

The method includes: transferring swine genetic material, e.g., a cellnucleus or a set of chromosomes, e.g. a complete set of chromosomes,which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, andDQ and in which at least 60% of all other genetic loci are homozygous,to a cell, wherein the cell is capable of developing into a swine,allowing the cell to develop into a swine, thereby providing a swinewhich is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, andDQ, and in which at least 60% of all other genetic loci are homozygous.

In preferred embodiments, the genetic material is transferred vianuclear transfer. For example, a swine cell nucleus, e.g., a nucleusfrom an undifferentiated swine cell, can be fused with a second cell,e.g., an oocyte, e.g., an enucleated oocyte, such as an enucleatedoocyte arrested in the metaphase of the second meiotic division, andthen transferred into a recipient swine, e.g., a maternal recipientswine. The embryo resulting from the fusion of the cell nucleus and theoocyte can also be cultured, e.g., cultured to the stage of blastocyst,and then transferred to the recipient swine.

In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore, of all other genetic loci in the swine are homozygous.

In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C,DR, and DQ can be of haplotype a (A^(a), B^(a), C^(a), DR^(a), DQ^(a)),haplotype c (A^(c), B^(c), C^(c), DR^(c), DQ^(c)), haplotype d (A^(d),B^(d), C^(d), DR^(d), DQ^(d)), haplotype g (A^(g), B^(g), C^(g), DR^(g),DQ^(g)), haplotype h (A^(h), B^(h), C^(h), DR^(h), DQ^(h)), or haplotypej (A^(j), B^(j), C^(j), DR^(j), DQ^(j)).

In another aspect, the invention features, a method of providing atransgenic swine, e.g., a transgenic miniature swine. The methodincludes:

providing a swine, e.g., a miniature swine described herein, which ishomozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and inwhich all other genetic loci are at least 60% homozygous; and

introducing a transgene into the swine, thereby preparing a transgenicswine.

In preferred embodiments the transgene encodes a xenogeneic, e.g., ahuman protein.

In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore, of all other genetic loci in the swine are homozygous.

In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C,DR, and DQ can be of haplotype a (A^(a), B^(a), C^(a), DR^(a), DQ^(a)),haplotype c (A^(c), B^(c), C^(c), DR^(c), DQ^(c)), haplotype d (A^(d),B^(d), C^(d), DR^(d), DQ^(d)), haplotype g (A^(g), B^(g), C^(g), DR^(g),DQ^(g)), haplotype h (A^(h), B^(h), C^(h), DR^(h), DQ^(h)), or haplotypej (A^(j), B^(j), C^(j), DR^(j), DQ^(j)).

In preferred embodiments, the swine is capable of reproduction, i.e.,the animal can produce functional gametes.

In another aspect, the invention features, a genetically engineeredswine cell, e.g., a cultured swine cell, a retrovirally transformedswine cell, or a cell derived from a transgenic swine, or purifiedpreparation of such cells, which include a transgene. The swine cell isfrom a swine, preferably a miniature swine, e.g., a miniature swine,which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, andDQ, and in which at least 60% of all other genetic loci are homozygous.

In preferred embodiments the transgene encodes a xenogeneic, e.g., ahuman protein.

In yet other preferred embodiments the genetically engineered swine cellis: a swine hematopoietic stem cell, e.g., a cord blood hematopoieticstem cell, a bone marrow hematopoietic stem cell, or a fetal or neonatalliver or spleen hematopoietic stem cell; derived from differentiatedblood cells, e.g. a myeloid cell, such as a megakaryocytes, monocytes,granulocytes, or an eosinophils; an erythroid cell, such as a red bloodcells, e.g. a lymphoid cell, such as B lymphocytes and T lymphocytes;derived from a pluripotent hematopoietic stem cell, e.g. a hematopoieticprecursor, e.g. a burst-forming units-erythroid (BFU-E), a colonyforming unit-erythroid (CFUE), a colony forming unit-megakaryocyte(CFU-Meg), a colony forming unit-granulocyte-monocyte (CFU-GM), a colonyforming unit-eosinophil (CFU-Eo), or a colony formingunit-granulocyte-erythrocyte-megakaryocyte-monocyte (CFU-GEMM); a swinecell other than a hematopoietic stem cell, or other blood cell; a swinethymic cell, e.g., a swine thymic stromal cell; a bone marrow stromalcell; a swine liver cell; a swine kidney cell; a swine epithelial cell;a swine hematopoietic progenitor cell; a swine muscle cell, e.g., aheart cell; or a dendritic cell or precursor thereof.

In yet other preferred embodiments the transgenic cell is: isolated orderived from cultured cells, e.g., a primary culture, e.g., a primarycell culture of hematopoietic stem cells; isolated or derived from atransgenic animal.

In yet other preferred embodiments: the transgenic swine cell ishomozygous for the transgene; the transgenic swine cell is heterozygousfor the transgene; the transgenic swine cell is homozygous for thetransgene (heterozygous transgenic swine can be bred to produceoffspring that are homozygous for the transgene); the transgenic swinecell includes two or more transgenes.

In another aspect, the invention features, a transgenic swine, e.g., aminiature swine, which is homozygous at swine leukocyte antigens (SLA)A, B, C, DR, and DQ, and in which at least 60% of all other genetic lociare homozygous and having cells which include a transgene.

In preferred embodiments the transgene encodes a xenogeneic, e.g., ahuman protein.

In yet other preferred embodiments the transgene includes a nucleic acidencoding a human peptide, e.g., a hematopoietic peptide, operably linkedto: a promoter other than the one it naturally occurs with; a swinepromoter, e.g., a swine hematopoietic gene promoter; a viral promoter,or an inducible or developmentally regulated promoter.

In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%,95% ormore, of all other genetic loci in the transgenic swine are homozygous.

In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C,DR, and DQ can be of haplotype a (A^(a), B^(a), C^(a), DR^(a), DQ^(a)),haplotype c (A^(c), B^(c), C^(c), DR^(c), DQ^(c)), haplotype d (A^(d),B^(d), C^(d), DR^(d), DQ^(d)), haplotype g (A^(g), B^(g), C^(g), DR^(g),DQ^(g)), haplotype h (A^(h), B^(h), C^(h), DR^(h), DQ^(h)), or haplotypej (A^(j), B^(j), C^(j), DR^(j), DQ^(j)).

In another aspect, the invention features, an isolated swine organ or aswine tissue from a transgenic swine, e.g., a miniature swine describedherein, which is homozygous at swine leukocyte antigens (SLA) A, B, C,DR, and DQ, and in which at least 60% of all other genetic loci arehomozygous, having cells which include a xenogeneic, e.g., a human,nucleic acid.

In preferred embodiments the organ is a heart, lung, kidney, pancreas,or liver.

In preferred embodiments the tissue is: thymic tissue; islet cells orislets; stem cells; bone marrow; endothelial cells; skin; or vasculartissue.

In another aspect, the invention features, a method of inducingtolerance in a recipient mammal of a first species, e.g., a human, to agraft from a donor mammal of a second species, e.g., a swine, forexample, a miniature swine described herein. The method includes:

providing a donor mammal, e.g., a miniature swine, which is from a herdwhich is homozygous for a major histocompatibility complex haplotype andat least 60% homozygous at all other genetic loci;

introducing into the recipient mammal, tolerance inducing tissue, e.g.,hematopoietic stem cells from the donor mammal, thymic tissue from thedonor mammal, or a nucleic acid which encodes an MHC antigen of thedonor mammal;

providing a graft from the donor mammal or from a second donor mammalfrom the herd; and introducing the graft into the recipient, therebyinducing tolerance in a recipient mammal of a first species to a graftfrom a mammal of the second species.

In another aspect, the invention features, a method of inducingtolerance in a recipient mammal of a first species, e.g., a human, to agraft from a donor mammal of a second species, e.g., a swine, forexample, a miniature swine described herein. The method includes:

providing a donor mammal, e.g., a miniature swine, which is from a herdwhich is homozygous for a major histocompatibility complex haplotype andat least 60% homozygous at all other genetic loci;

introducing into the recipient mammal, hematopoietic stem cells from thedonor mammal;

providing a graft from the donor mammal or from a second donor mammalfrom the herd; and

introducing the graft into the recipient, thereby inducing tolerance ina recipient mammal of a first species to a graft from a mammal of thesecond species.

In preferred embodiments, the recipient is a primate and the donor is aswine, e.g., a miniature swine; the recipient is a human and the donoris a swine, e.g., a miniature swine.

In preferred embodiments, the donor is a swine, preferably a miniatureswine, which is homozygous at swine leukocyte antigens (SLA) A, B, C,DR, and DQ, and is from a herd which at least 60% of all other geneticloci are homozygous. In other preferred embodiments, at least 65%, 70%,75%, 80%, 85%, 90%, 95% or more, of all other genetic loci in the swineare homozygous.

In preferred embodiments, the donor is a swine and the swine leukocyteantigens (SLA) A, B, C, DR, and DQ can be of haplotype a (A^(a), B^(a),C^(a), DR^(a), DQ^(a)), haplotype c (A^(c), B^(c), C^(c), DR^(c),DQ^(c)), haplotype d (A^(d), B^(d), C^(d), DR^(d), DQ^(d)), haplotype g(A^(g), B^(g), C^(g), DR^(g), DQ^(g)), haplotype h (A^(h), B^(h), C^(h),DR^(h), DQ^(h)), or haplotype j (A^(j), B^(j), C^(j), DR^(j), DQ^(j)).

In preferred embodiments the method is practiced without T celldepletion, e.g., without the administration of thymic irradiation, or Tcell depleting anti T cell antibodies.

In preferred embodiments the method includes: administering to therecipient, one or both, of an inhibitor, e.g., a blocker, of the CD40ligand-CD40 interaction and a blocker of the CD28-B7 interaction. TheCD40 ligand-CD40 pathway can be inhibited by administering an antibodyor soluble receptor for the CD40 ligand or CD40, e.g., by administeringCTLA4-1gG. Preferably the inhibitor binds the CD40 ligand. The CD28-B7pathway can be inhibited by administering a soluble receptor or antibodyfor the CD28 or B7, e.g., an anti-B7 antibody. Preferably, the inhibitorbinds B7. In preferred embodiments CTLA4-1gG and an anti-b7 antibody areadministered.

In preferred embodiments the method can be practiced without theadministration of hematopoietic space-creating irradiation, e.g., wholebody irradiation.

In preferred embodiments the method includes administering asufficiently large number of donor hematopoietic cells to the recipientsuch that, donor stem cells engraft, give rise to mixed chimerism, andinduce tolerance without space-creating treatment. In preferredembodiments the number of donor hematopoietic cells is at least twice,is at least equal to, or is at least 75, 50, or 25% as great as, thenumber of bone marrow cells found in an adult of the recipient species.In preferred embodiments the number of donor hematopoietic stem cells isat least twice, is at least equal to, or is at least 75, 50, or 25% asgreat as, the number of bone marrow hematopoietic stem cells found in anadult of the recipient species. In the case where an inbred populationof the donor species exists, e.g., where the donor species is miniatureswine, the number of available donor cells is not limited to the numberof cells which can be obtained from a single animal. Thus, in suchcases, the donor cells administered to the recipient can come from morethan one, e.g., from two, three, four, or more animals.

The number of donor cells administered to the recipient can be increasedby either increasing the number of stem cells provided in a particularadministration or by providing repeated administrations of donor stemcells.

Repeated stem cell administration can promote engraftment, mixedchimerism, and long-term deletional tolerance in graft recipients. Thus,the invention also includes methods in which multiple hematopoietic stemcell administrations are provided to a recipient. Multipleadministration can substantially reduce or eliminate the need forhematopoietic space-creating irradiation. Administrations can be givenprior to, at the time of, or after graft implantation. In preferredembodiments multiple administrations of stem cells are provided prior tothe implantation of a graft. Two, three, four, five, or moreadministrations can be provided. The period between administrations ofhematopoietic stem cells can be varied. In preferred embodiments asubsequent administration of hematopoietic stem cell is provided: atleast two days, one week, one month, or six months after the previousadministration of stem cells; when the recipient begins to show signs ofhost lymphocyte response to donor antigen; when the level of chimerismdecreases; when the level of chimerism falls below a predeterminedvalue; when the level of chimerism reaches or falls below a level wherestaining with a monoclonal antibody specific for a donor PBMC antigen isequal to or falls below staining with an isotype control which does notbind to PBMC's, e.g. when the donor specific monoclonal stains less than1-2% of the cells; or generally, as is needed to maintain a level ofmixed chimerism sufficient to maintain tolerance to donor antigen.

When multiple stem cell administrations are given, one or more of theadministrations can include a number of donor hematopoietic cells whichis at least twice, is equal to, or is at least 75, 50, or 25% as greatas, the number of bone marrow cells found in an adult of the recipientspecies; include a number of donor hematopoietic stem cells which is atleast twice, is equal to, or is at least 75, 50, or 25% as great as, thenumber of bone marrow hematopoietic stem cells found in an adult of therecipient species.

Although the methods described herein, e.g., those in which blockers ofboth pathways are administered, or those in which a relatively largenumber of hematopoietic stem cells are administered, will ofteneliminate the need for other preparative steps, some embodiments includeinactivating preferably graft reactive or xenoreactive, e.g., swinereactive, NK cells, of the recipient mammal. This can be accomplished,e.g., by introducing into the recipient mammal an antibody capable ofbinding to natural killer cells of the recipient mammal. Theadministration of antibodies, or other treatment to inactivate naturalkiller cells, can be given prior to introducing the hematopoietic stemcells into the recipient mammal or prior to implanting the graft in therecipient. This antibody can be the same or different from an antibodyused to inactivate T cells.

Although the methods described herein, e.g., those in which blockers ofboth pathways are administered, or those in which a relatively largenumber of hematopoietic stem cells are administered, will ofteneliminate the need for other preparative steps, some embodiments includeinactivating e.g., by depleting natural killer cells, T cells,preferably graft reactive or xenoreactive, e.g., swine reactive, T cellsof the recipient mammal. This can be accomplished, e.g., by introducinginto the recipient mammal an antibody capable of binding to T cells ofthe recipient mammal. The administration of antibodies, or othertreatment to inactivate T cells, can be given prior to introducing thehematopoietic stem cells into the recipient mammal or prior toimplanting the graft in the recipient. This antibody can be the same ordifferent from an antibody used to inactivate natural killer cells.

Other preferred embodiments include: the step of introducing into therecipient mammal, donor species-specific stromal tissue, preferablyhematopoietic stromal tissue, e.g., fetal liver or thymus. In preferredembodiments: the stromal tissue is introduced simultaneously with, orprior to, the hematopoietic stem cells; the hematopoietic stem cells areintroduced simultaneously with, or prior to, the antibody.

Although the methods described herein, e.g., those in which blockers ofboth pathways are administered, or those in which a relatively largenumber of hematopoietic.stem cells are administered, will ofteneliminate the need for other preparative steps, some embodiments include(optionally): the step of, prior to hematopoietic stem celltransplantation, creating hematopoietic space, e.g., by irradiating therecipient mammal with low dose, e.g., less than 400, preferably lessthan 300, more preferably less than 200 or 100 rads, whole body,irradiation to deplete or partially deplete the bone marrow of therecipient. As is discussed herein this treatment can be reduced orentirely eliminated.

Other preferred embodiments include: the step of, preferably prior tohematopoietic stem cell transplantation, depleting natural antibodiesfrom the blood of the recipient mammal. Depletion can be achieved, byway of example, by contacting the recipients blood with an epitope whichabsorbs performed anti-donor antibody. The epitope can be coupled to aninsoluble substrate and provided, e.g., as an affinity column. E.g., anα1-3 galactose linkage epitope-affinity matrix, e.g., matrix boundlinear B type VI carbohydrate, can be used to deplete naturalantibodies. Depletion can also be achieved by hemoperfusing an organ,e.g., a liver or a kidney, obtained from a mammal of the donor species.(In organ hemoperfusion antibodies in the blood bind to antigens on thecell surfaces of the organ and are thus removed from the blood). Otherpreferred embodiments include those in which: the same mammal of thesecond species is the donor of one or both the graft and thehematopoietic cells.

In preferred embodiments, the method includes the step of introducinginto the recipient a graft, obtained from the donor which is obtainedfrom a different organ than the hematopoietic stem cells, e.g., a heart,pancreas, liver, or kidney.

In preferred embodiments the host or recipient is a post-natalindividual, e.g., an adult, or a child.

In preferred embodiments the method further includes the step ofidentifying a host or recipient which is in need of a graft.

In another aspect, the invention features a method of inducing tolerancein a recipient mammal of a first species, e.g., a human, to a graft froma donor mammal of a second species, e.g., a swine, for example, aminiature swine. The method includes:

providing a donor mammal which is from a herd which is homozygous for amajor histocompatibility complex haplotype and at least 60% homozygousat all other genetic loci;

introducing into the recipient mammal, thymic tissue from the donormammal;

providing a graft from the donor mammal, or from a second donor mammalfrom the herd; and

introducing the graft into the recipient, thereby inducing tolerance ina recipient mammal of a first species to a graft from a mammal of thesecond species.

In preferred embodiments, the recipient is a primate and the donor is aswine, e.g., a miniature swine; the recipient is a human and the donoris a swine, e.g., a miniature swine.

In preferred embodiments, the donor is a swine and is from a herd whichis homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, andis from a herd in which at least 60% of all other genetic loci arehomozygous. In other preferred embodiments, at least 65%, 70%, 75%, 80%,85%, 90%, 95% or more, of all other genetic loci in the swine herd arehomozygous.

In preferred embodiments, the donor is a swine and the swine leukocyteantigens (SLA) A, B, C, DR, and DQ can be of haplotype a (A^(a), B^(a),C^(a), DR^(a), DQ^(a)), haplotype c (A^(c), B^(c), C^(c), DR^(c),DQ^(c)), haplotype d (A^(d), B^(d), C^(d), DR^(d), DQ^(d)), haplotype g(A^(g), B^(g), C^(g), DR^(g), DQ^(g)), haplotype h (A^(h), B^(h), C^(h),DR^(h), DQ^(h)), or haplotype j (A^(j), B^(j), C^(j), DR^(j), D^(j)).

In preferred embodiments the method is practiced without T celldepletion or inactivation, e.g., without the administration of thymicirradiation, or T cell depleting anti T cell antibodies.

In preferred embodiments the method includes: administering to therecipient, one or both, of an inhibitor, e.g., a blocker, of the CD40ligand-CD40 interaction and a blocker of the CD28-B7 interaction. TheCD40 ligand-CD40 pathway can be inhibited by administering an antibodyor soluble receptor for the CD40 ligand or CD40, e.g., by administeringCTLA4-1gG. Preferably the inhibitor binds the CD40 ligand. The CD28-B7pathway can be inhibited by administering a soluble receptor or antibodyfor the CD28 or B7, e.g., an anti-B7 antibody. Preferably, the inhibitorbinds B7. In preferred embodiments CTLA4-1gG and an anti-b7 antibody areadministered.

Although the methods described herein, e.g., those in which blockers ofboth pathways are administered, will often eliminate the need for otherpreparative steps, some embodiments include inactivating natural killercells, preferably graft reactive or xenoreactive, e.g., swine reactive,NK cells, of the recipient mammal. This can be accomplished, e.g., byintroducing into the recipient mammal an antibody capable of binding tonatural killer cells of the recipient mammal. The administration ofantibodies, or other treatment to inactivate natural killer cells, canbe given prior to introducing the thymic tissue into the recipientmammal or prior to implanting the graft in the recipient. This antibodycan be the same or different from an antibody used to inactivate Tcells.

Although methods described herein, e.g., those in which blockers of bothpathways are administered, will often eliminate the need for otherpreparative steps, some embodiments include inactivating, e.g., ydepleting T cells, preferably graft reactive or xenoreactive, e.g.,swine reactive, T cells of the recipient mammal. This can beaccomplished, e.g., by introducing into the recipient mammal an antibodycapable of binding to T cells of the recipient mammal. Theadministration of antibodies, or other treatment to inactivate T cells,can be given prior to introducing the thymic tissue into the recipientmammal or prior to implanting the graft in the recipient. This antibodycan be the same or different from an antibody used to inactivate naturalkiller cells.

Other preferred embodiments include: the step of, preferably prior tothymic tissue transplantation, depleting natural antibodies from theblood of the recipient mammal. Depletion can be achieved, by way ofexample, by contacting the recipients blood with an epitope whichabsorbs performed anti-donor antibody. The epitope can be coupled to aninsoluble substrate and provided, e.g., as an affinity column. E.g., anα1-3 galactose linkage epitope-affinity matrix, e.g., matrix boundlinear B type VI carbohydrate, can be used to deplete naturalantibodies. Depletion can also be achieved by hemoperfusing an organ,e.g., a liver or a kidney, obtained from a mammal of the donor species.(In organ hemoperfusion antibodies in the blood bind to antigens on thecell surfaces of the organ and are thus removed from the blood.)

Other preferred embodiments include those in which: the same mammal ofthe second species is the donor of one or both the graft and the thymictissue.

In preferred embodiments, the method includes the step of introducinginto the recipient a graft obtained from the donor which is obtainedfrom a different organ than the thymic tissue, e.g., a heart, pancreas,liver, or kidney.

In preferred embodiments the host or recipient is a post-natalindividual, e.g., an adult, or a child.

In preferred embodiments the method further includes the step ofidentifying a host or recipient which is in need of a graft.

In another aspect, the invention features a method of inducing tolerancein a recipient mammal, preferably a primate, e.g., a human, to a graftobtained from a donor mammal of a second species, e.g., a swine, e.g., aminiature swine, which graft preferably expresses an MHC antigen.

The Method Includes:

inserting a nucleic acid, e.g., DNA, encoding an MHC antigen into ahematopoietic stem cell, e.g., bone marrow hematopoietic stem cell, ofthe recipient, wherein the nucleic acid encodes an MHC antigen of aswine, e.g., a miniature swine, from a herd which is homozygous for amajor histocompatibility complex haplotype and at least 60% o homozygousat all other genetic loci; allowing the MHC antigen encoding nucleicacid to be expressed in the recipient; and preferably, implanting thegraft in the recipient, wherein the graft is from an animal from theherd.

In preferred embodiments, the donor is a swine from a herd which ishomozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and inwhich at least 60% of all other genetic loci are homozygous. In otherpreferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore, of all other genetic loci in the swine are homozygous.

In preferred embodiments, the donor is a swine and the swine leukocyteantigens (SLA) A, B, C, DR, and DQ can be of haplotype a (A^(a), B^(a),C^(a), DR^(a), DQ^(a)), haplotype c (A^(c), B^(c), C^(c), DR^(c),DQ^(c)), haplotype d (A^(d), B^(d), C^(d), DR^(d), DQ^(d)), haplotype g(A^(g), B^(g), C^(g), DR^(g), DQ^(g)), haplotype h (A^(h), B^(h), C^(h),DR^(h), DQ^(h)), or haplotype j (A^(j), B^(j), C^(j), DR^(j), DQ^(j)).

Preferred embodiments include those in which: the cell is removed fromthe recipient prior to the nucleic acid insertion and returned to therecipient after the nucleic acid insertion; the nucleic acid includes aMHC class I gene, e.g., a (SLA) A, B, C gene; the nucleic acid includesa MHC class II gene, e.g., a DR or DQ gene; the nucleic acid is insertedinto the cell by transduction, e.g. by a retrovirus, e.g., by aMoloney-based retrovirus; and the nucleic acid is expressed in bonemarrow cells and/or peripheral blood cells of the recipient at least 14,preferably 30, more preferably 60, and most preferably 120 days, afterthe nucleic acid is introduced into the recipient.

In preferred embodiments the method is practiced without T celldepletion, e.g., without the administration of thymic irradiation, or Tcell depleting anti T cell antibodies.

In preferred embodiments the method includes: administering to therecipient, one or both, of an inhibitor, e.g., a blocker, of the CD40ligand-CD40 interaction and a blocker of the CD28-B7 interaction. TheCD40 ligand-CD40 pathway can be inhibited by administering an antibodyor soluble receptor for the CD40 ligand or CD40, e.g., by administeringCTLA4-1gG. Preferably the inhibitor binds the CD40 ligand. The CD28-B7pathway can be inhibited by administering a soluble receptor or antibodyfor the CD28 or B7, e.g., an anti-B7 antibody. Preferably, the inhibitorbinds B7. In preferred embodiments CTLA4-1gG and an anti-b7 antibody areadministered.

Although the methods described herein, e.g., those in which blockers ofboth pathways are administered, will often eliminate the need for otherpreparative steps, some embodiments include inactivating natural killercells, preferably graft reactive or xenoreactive, e.g., swine reactive,NK cells, of the recipient mammal. This can be accomplished, e.g., byintroducing into the recipient mammal an antibody capable of binding tonatural killer cells of the recipient mammal. The administration ofantibodies, or other treatment to inactivate natural killer cells, canbe given prior to introducing the hematopoietic stem cells into therecipient mammal or prior to implanting the graft in the recipient. Thisantibody can be the same or different from an antibody used toinactivate T cells.

Although the methods described herein, e.g., those in which blockers ofboth pathways are administered, will often eliminate the need for otherpreparative steps, some embodiments include inactivating T cells,preferably graft reactive or xenoreactive, e.g. swine reactive, T cellsof the recipient mammal. This can be accomplished, e.g., by introducinginto the recipient mammal an antibody capable of binding to T cells ofthe recipient mammal. The administration of antibodies, or othertreatment to inactivate, e.g., deplete, T cells, can be given prior tointroducing the hematopoietic stem cells into the recipient mammal orprior to implanting the graft in the recipient. This antibody can be thesame or different from an antibody used to inactivate natures killercells.

Preferred embodiments include (optionally): the step of, prior toengineered hematopoietic stem cell transplantation, creatinghematopoietic space, e.g., by irradiating the recipient mammal with lowdose, e.g., less than 400, preferably less than 300, more preferablyless than 200 or 100 rads, whole body irradiation to deplete orpartially deplete the bone marrow of the recipient.

Other preferred embodiments include: the step of, preferably prior toengineered hematopoietic stem cell transplantation, depleting naturalantibodies from the blood of the recipient mammal. Depletion can beachieved, by way of example, by contacting the recipients blood with anepitope which absorbs performed anti-donor antibody. The epitope can becoupled to an insoluble substrate and provided, e.g., as an affinitycolumn. E.g., an α1-3 galactose linkage epitope-affinity matrix, e.g.,matrix bound linear B type VI carbohydrate, can be used to depletenatural antibodies. Depletion can also be achieved by hemoperfusing anorgan, e.g., a liver or a kidney, obtained from a mammal of the donorspecies. (In organ hemoperfusion antibodies in the blood bind toantigens on the cell surfaces of the organ and are thus removed from theblood.)

In preferred embodiments, the method includes the step of introducinginto the recipient a graft obtained from the donor which is obtainedfrom a different organ than the hematopoietic stem cells, e.g., a heart,pancreas, liver, or kidney.

In preferred embodiments the host or recipient is a post-natalindividual, e.g., an adult, or a child.

In preferred embodiments the method further includes the step ofidentifying a host or recipient which is in need of a graft.

The retroviral methods of the invention allow the reconstitution of agraft recipient's bone marrow with transgenic autologous bone marrowcells expressing a donor MHC gene. Expression of a transgenic MHC geneconfers tolerance to grafts which exhibit the products of these orclosely related MHC genes. Thus, these methods provide for the inductionof specific transplantation tolerance by somatic transfer of MHC genes.Retroviral methods of the invention avoid the undesirable side effectsof broad spectrum immune suppressants which are often used intransplantation.

In another aspect, the invention features, a method of selectivelybreeding animals described herein to improve or maintain fecundity of aherd. The method includes:

mating a first sow of a herd with a mate from the herd;

mating a second sow of the herd with the same or a different male fromthe herd; determining which sow has higher fecundity;

mating the sow with the highest fecundity (or an offspring of said sow)to thereby improve or maintain fecundity of the herd.

A herd of the invention can be expanded by matings between males andfemales drawn from the herd. The zygotes which result from such matingscan be allowed to develop in the female which produced the egg or eggswhich were fertilized in the mating. The herd can also be expanded byimplanting a zygote (wherein the zygote produced by the union of a spermcell produced by a male of the herd with an egg produced by a female ofthe herd) in a foster mother. The foster mother can be from the herd orcan be an animal which is not from the herd. For example, a “herd”zygote can be implanted in an outbred foster mother. This method canallow for rapid expansion of a herd. Accordingly, in another aspect, theinvention features a method of expanding an inbred herd, e.g., a herddescribed herein. The method includes:

providing a zygote which is produced by the union of a sperm cellproduced by a male of the herd with an egg produced by a female of theherd;

implanting the zygote into a foster mother, e.g., a female which ispreferably not from the herd, allowing the zygote to give rise to aninbred swine, thereby expanding the herd.

“A preparation of cells”, as used herein, refers to cells which arephysically separated from the animal which produces them.

“An isolated nucleus”, as used herein, refers to a nucleus which hasbeen removed from the cell of its origin.

“An isolated organ”, as used herein, refers to an organ or tissue whichhas been physically separated from the animal which produces it.

“A hematopoietic stem cell preparation, as used herein, is a populationof cells which includes hematopoietic stem cells. The preparation can bepure, or it can include other cell types.” A juvenile miniature swine,is a swine which has not reached sexual maturity.

“An adult miniature swine” is one which has reached sexual maturity.

“A herd,” as used herein, refers to a group of at least one male and onefemale which can breed to produce fertile male and female offspring. Allof the animals of a herd are homozygous at SLA loci: A, B, C, DR and DQ,and all animals in the herd are homozygous for the same allele at SLA A,B, C, DR and DQ. Thus, only one allele for each of SLA A, B, C, DR, orDQ is present in the herd. Furthermore, the herd is highly inbred at allother loci. At least 60%, and preferably at least 65%, 70%, 75%, 80%,85%, 90%, 95%, of all other loci are homozygous and for each of theirloci, all swine in the herd are homozygous for the same allele. Thus ina herd wherever at least 85% of the loci are homozygous, there is nogenetic variation in the herd for at least 85% of the loci. Homozygositycan be determined, e.g., by minisatellite analysis or mathematically.

“Graft”, as used herein, refers to a body part, organ, tissue, or cells.Organs such as liver, kidney, heart or lung, or other body parts, suchas bone or skeletal matrix, tissue, such as skin, intestines, endocrineglands, or progenitor stem cells of various types, are all examples ofgrafts. “Hematopoietic stem cell”, as used herein, refers to a cell,e.g., a bone marrow cell, or a fetal liver or spleen cell, which iscapable of developing into all myeloid and lymphoid lineages and byvirtue of being able to self-renew can provide long term hematopoieticreconstitution. Preparations of hematopoietic cells or preparations,such as bone marrow, which include other cell types, can be used inmethods of the invention. Although not wishing to be bound by theory, itis believed that the hematopoietic stem cells home to a site in therecipient mammal. The preparation should include immature cells, i.e.,undifferentiated hematopoietic stem cells; these desired cells can beseparated out of a preparation or a complex preparation can beadministered. E.g., in the case of bone marrow stem cells, the desiredprimitive cells can be separated out of a preparation or a complex bonemarrow sample including such cells can be used. Hematopoietic stem cellscan be from fetal, neonatal, immature or mature animals. Stem cellsderived from the cord blood of the recipient or the donor can be used inmethods of the invention. See U.S. Pat. No. 5,192,553, herebyincorporated by reference, and U.S. Pat. No. 5,004,681, herebyincorporated by reference.

“Thymic or lymph node or thymocytes or T cell”, as used herein, refersto thymocytes or T cells which are resistant to inactivation bytraditional methods of T cell inactivation, e.g., inactivation by asingle intravenous administration of anti-T cell antibodies, e.g.,antibodies, e.g., ATG preparation.

“Thymic irradiation”, as used herein, refers to a treatment in which atleast half, and preferably at least 75, 90, or 95% of the administeredirradiation is targeted to the thymus. Whole body irradiation, even ifthe thymus is irradiated in the process of delivering the whole tobodyirradiation, is not considered thymic irradiation.

“MHC antigen”, as used herein, refers to a protein product of one ormore MHC genes; the term includes fragments or analogs of products ofMHC genes which can evoke an immune response in a recipient organism.Examples of MHC antigens include the products (and fragments or analogsthereof) of the human MHC genes, i.e., the HLA genes. MHC antigens inswine, e.g., miniature swine, include the products (and fragments andanalogs thereof) of the SLA genes, e.g., the DRB gene.

“Hematopoietic space-creating irradiation”, as used herein, refers toirradiation directed to the hematopoietic tissue, i.e., to tissue inwhich stem cells are found, e.g., the bone marrow. It is of sufficientintensity to kill or inactivate a substantial number of hematopoieticcells. It is often given as whole body irradiation.

“Thymic space” as used herein, is a state created by a treatment thatfacilitates the migration to and/or development in the thymus of donorhematopoietic cells of a type which can delete or inactivate hostthymocytes that recognize donor antigens. It is believed that the effectis mediated by elimination of host cells in the thymus.

“Stromal tissue”, as used herein, refers to the supporting tissue ormatrix of an organ, as distinguished from its functional elements orparenchyma.

“Tolerance”, as used herein, refers to an inhibition of a graftrecipient's immune response which would otherwise occur, e.g., inresponse to the introduction of a non-self MHC antigen into therecipient. Tolerance can involve humoral, cellular, or both humoral andcellular responses. Tolerance, as used herein, refers not only tocomplete immunologic tolerance to an antigen, but to partial immunologictolerance, i.e. a degree of tolerance to an antigen which is greaterthan what would be seen if a method of the invention were not employed.Tolerance, as used herein, refers to a donor antigen-specific inhibitionof the immune system as opposed to the broad spectrum inhibition of theimmune system seen with immunosuppressants.

“A blocker” as used herein, refers to a molecule which binds a member ofa ligand/counter-ligand pair and inhibits the interaction between theligand and counter-ligand or which disrupts the ability of the boundmember to transduce a signal. The blocker can be an antibody (orfragment thereof) to the ligand or counter ligand, a soluble ligand(soluble fragment of the counter ligand), a soluble counter ligand(soluble fragment of the counter ligand), or other protein, peptide orother molecule which binds specifically to the counter-ligand or ligand,e.g., a protein or peptide selected by virtue of its ability to bind theligand or counter ligand in an affinity assay, e.g., a page displaysystem.

The term “haplotype” as used herein refers to a group of alleles fromclosely linked loci which are usually inherited as a unit. For example,in the MHC locus in swine the SLAa haplotype codes for the SLA-A^(a),B^(a), C^(a), DR^(a), and DQ^(a) alleles, the SLAd haplotype codes forthe SLA-A^(d), B^(d), C^(d), DR^(d), and DQ^(d) alleles, etc.

The terms “organ” and “tissue” as used herein, mean any biologicalmaterial that is capable of being transplanted and include organs(especially the internal vital organs such as the heart, lung, liver,kidney, pancreas and thyroid), cornea, skin, blood vessels and otherconnective tissue, cells including blood and hematopoietic cells, Isletsof Langerhans, brain cells and cells from endocrine and other organs andbodily fluids, all of which may be candidate for transplantation.

As used herein, the term “transgene” refers to a nucleic acid sequence(encoding, e.g., one or more class I or class II MHC proteins), which ispartly or entirely heterologous, i.e., foreign, to the transgenic animalor cell into which it is introduced or which when introduced into thegenome results in a change of sequence in the genome. A transgene caninclude one or more transcriptional regulatory sequences and any othernucleic acid, such as introns, that may be necessary for optimalexpression of the selected nucleic acid, all operably linked to theselected nucleic acid, and may include an enhancer sequence.

As used herein, a “transgenic swine” is any swine which is homozygous atswine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which atleast 60% of all other genetic loci are homozygous, and in which one ormore, and preferably essentially all, of the cells of the animal includea transgene. The transgene can be introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. The term genetic manipulation doesnot include classical cross-breeding, or in vitro fertilization, butrather is directed to the introduction of a recombinant DNA molecule.This molecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA.

As used herein, the term “genetically engineered swine cells” refers tocells derived from a swine which is homozygous at swine leukocyteantigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of allother genetic loci are homozygous, and which have been used asrecipients for a recombinant vector or other transfer nucleic acid, andinclude the progeny of the original cell which has been transfected ortransformed. Genetically engineered swine cells include cells in whichtransgenes or other nucleic acid vectors have been incorporated into thehost cell's genome, as well as cells harboring expression vectors whichremain autonomous from the host cell's genome.

As used herein, the term “propagatable” refers to animals which arecapable of giving rise to viable offspring by sexual or asexualreproduction. Preferably, animals of the invention are propagatable.

The high degree of genetic uniformity characteristic of animalsdescribed herein allows for considerable advantages in terms of qualityassurance. For example, any single animal is representative of the herd,i.e., the same or very similar (allow for differences of are or gender)to any other, in terms of immunogenetics, size, physiology, and health.

Genetically uniform animals described herein are useful geneticengineering, for example, a first modification, e.g., the introductionof a first transgene can be made in a first animal. A secondmodification, e.g., the introduction of a different second transgene,can be made in a second animal. The appropriate matings can be performedto yield an animal having both modifications. Except for themodifications, all of the modified animals, as well as non-modifiedanimals of the herd, are highly uniform. Thus, genetically engineeredmodifications can be introduced by matings between modified animals,with minimal introduction of changes in the genetic background.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION

The drawings are first briefly described.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a depiction of a time course of cytotoxic antibody formation.

FIG. 2 is a schematic representation of the serological analysis of thefirst generation progeny. An analysis of the progeny for MHC genotypesby absorption of cytotoxic antisera is shown schematically for the caseof maximum possible heterogeneity. Experimental details are provided inSachs, et al. (1976) Transplantation 22:559-567.

FIG. 3 is a schematic representation of the breeding scheme employed inthe production of MSLA homozygous miniature swine.

FIG. 4 is a depiction of hybridization of RT-PCR Amplified ReactionProducts with a PERV Specific Oligonucleotide: Day 42 Cell FreeSupernatants. Lane # Sample:

-   1. DNA size markers;-   2. No template control;-   3. Negative control;-   4. Negative control;-   5. 293→293;-   6. 293→293;-   7. ST Iowa→293;-   8. ST Iowa→293;-   9. Non-irradiated PBMCs/293→293;-   10. Non-irradiated PBMCs/293→293;-   11. Irradiated PBMCs/293→293;-   12. Irradiated PBMCs/293→293;-   13. Non-irradiated PBMCs/ST Iowa→293;-   14. Non-irradiated PBMCs/ST Iowa→293;-   15. Irradiated PBMCs/ST Iowa→293;-   16. Irradiated PBMCs/ST Iowa→293;-   17. Empty;-   18. Empty;-   19. 2700 PERV virus particles;-   20. Empty;-   21. DNA size markers;-   22. PK-15 monoculture→293;-   23. PK-15 monoculture→293;-   24. 293 inoculated with PERV→293;-   25. 293 inoculated with PERV→293;-   26. Irradiated PK-15/293→293;-   27. Irradiated PK-15/293→293;-   28. Irradiated PK-15/293→293;-   29. Irradiated PK-15/293→293;-   30. Empty;-   31. Empty;-   32. 293→293 spiked with 27;00 vp;-   33. 293→293 spiked with 2700 vp;-   34. 293→293 spiked with 270 vp;-   35. 293→293 spiked with 270 vp;-   36. Empty;-   37. 2700 PERV virus particles;-   38. 270 PERV virus particles;-   39. 27 PERV virus particles;-   Empty

Inbred Miniature Swine

Many important advances in modern transplantation immunology have beenmade possible by the availability of inbred strains of mice and rats.The production of such strains involves sequential brother-sister matingfor more than 20 generations, by which time >98% of segregating lociwould be expected to have become fixed, that is, homozygous for one ofthe four possible alleles that might have been present at the time ofthe first brother-sister mating. As such, animals of an inbred strainare essentially identical to one another, i.e., are genetically similarto identical twins. From an experimental viewpoint, this removes many ofthe genetic factors that lead to heterogeneity of experimental results,making it possible to evaluate more accurately the effect of specifictreatments on the course of biologic phenomena. In the case oftransplantation biology, the availability of such inbred strains madepossible the discovery of the laws of transplantation and permitted theidentification and characterization of numerous transplantationantigens.

Despite the enormous usefulness of mice for studies of transplantation,there are a variety of areas of research, especially preclinicalresearch, in which large animals have advantages over rodent models.These advantages have practical importance, such as meeting sizerequirements for some surgical transplantation procedures. They alsohave theoretical importance in terms of similarity to humans inphysiologic and immunologic characteristics. However, true inbreedingwould not be feasible for most large animal species within a reasonableperiod of time, since the minimum time necessary for 20 sequentialpedigreed brother-sister matings is approximately 7 years for mice butwould range from 30-200 years for the commonest large experimentalanimals. In addition, during the process of inbreeding many strains arelost due to the fixation of recessive lethal mutations.

For the purposes of transplantation biology, it is clear that the majorhistocompatibility complex is of overwhelming importance in determiningthe outcome of transplants. Therefore, the decision was made to producea large animal model consisting of partially inbred animals homozygousfor different alleles at the MHC. For this purpose, a selectivepedigreed inbreeding scheme was used in which breeders were selected onthe basis of characteristics attributable to the MHC.

Miniature swine, in particular, exhibit several attractivecharacteristics:

-   1. Breeding characteristics. Like their domestic counterparts,    miniature swine reach sexual maturity at an age of 4-5 months, more    preferably 6-7 months. They give birth to multiple offspring (3-10    per litter), making it possible to select appropriate animals at    each generation. In addition, they have an estrous cycle every 3    weeks, permitting breeding throughout the year.-   2. Similarity to humans. Miniature swine reach an adult size of    200-300 pounds, in contrast to domestic swine that attain weights of    over 1000 pounds and are therefore unmanageable as laboratory    animals. The size of miniature swine makes it possible to study    animals of weights approximately equivalent to that of human beings.    Many aspects of the porcine immune system are also very similar to    that of human beings and swine lymphocytes can generally be treated    by procedures identical to those optimized for human studies. In    addition, swine are physiologically similar to humans and have been    an important model for cardiovascular research.

Accordingly, utilizing a selective inbreeding scheme, a herd ofpartially inbred miniature swine has been developed, in which the MHCequivalent-termed swine leukocyte antigen (SLA) in swine—has been fixedfor three alleles. The initial breeders were chosen from different,independently established herds of miniature swine. The initial boar waspurchase from Vita Vet Laboratories, Marion, Ind. and the initial sowwas a “pigmee pig” purchased from the Hormel Institute, Austin, Tex.

Further breeding of the MHC inbred herds by brother-sister matingswithin each of the herd was performed as described in Sachs, et al.(1976) Transplantation 22:559-567 the contents of which are incorporatedherein by reference, in an attempt to derive true inbred lines.

Transgenic Swine

Swine, cells, tissues, organs and other compositions of the inventioncan include a transgene.

In preferred embodiments the transgene encodes a xenogeneic, e.g., ahuman protein, e.g., a class I MHC protein, e.g., an HLA A, B, C or Ggene. The inclusion of a xenogeneic class I gene can be used to prolongacceptance of a graft, as is described in U.S. Ser. No. 08/692,843,filed Aug. 2, 1996, and hereby incorporated by reference.

In preferred embodiments the transgene includes an a subunit, e.g., anHLA class I gene, e.g., an HLA C gene.

Where the transgene includes an HLA C gene, the allele, by way ofexample, can be any Cw1, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7, Cw8, Cw9, Cw7/8v,or Cw10 allele. Alleles of HLA class I genes can often be classed intoreactivity groups wherein an allele from a reactivity group can conferprotection against NK cells specific to other alleles in the reactivitygroup. Thus, in preferred embodiments, the transgene includes an allelewhich is a member of a reactivity group, e.g., a Group 1 allele, e.g.,any of an HLA C Cw2, Cw4, Cw5, or Cw6 allele, or a Group 2 allele, e.g.,any of an HLA C Cwl1, Cw3, Cw7, or Cw8 allele. In other preferredembodiments the allele has: an Asn at residue 77 and a Lys at residue80; or a Ser at residue 77 and an Asn at residue 80.

In preferred embodiments the transgene includes an HLA A gene. In otherpreferred embodiments the transgene includes an HLA B gene.

In other preferred embodiments the transgene includes an HLA G gene,e.g., any of alleles I-IV of HLA G.

In preferred embodiments: the transgenic swine cell, tissue or organ,includes, in addition to the first transgene, a second transgene whichincludes a class I MHC protein. In preferred embodiments the secondtransgene includes an HLA class I gene, e.g., an HLA A, B, C or G gene.In preferred embodiments the first transgene includes an allele from afirst reactivity group and the second transgene includes an allele froma second reactivity group. For example, the first transgene includes aGroup 1 allele, e.g., any of an HLA C Cw2, Cw4, Cw5, or Cw6 allele, andthe second transgene includes a Group 2 allele, e.g., any of an HLA CCwl, Cw3, Cw7, or Cw8 allele. In preferred embodiments the firsttransgene encodes an allele which has an Asn at residue 77 and a Lys atresidue 80 and the second transgene encodes an allele which has a Ser atresidue 77 and an Asn at residue 80. In other preferred embodiments thesecond transgene encodes a human β subunit; e.g., a β-2 microglobulingene.

In preferred embodiments the transgene includes a chimeric class I gene,e.g., a chimeric HLA A, B, C, or G gene. The chimeric transgene caninclude a first portion derived from a first allele of a gene encoding aclass I protein and a second portion derived from a second allele of thegene encoding the class I protein. In other embodiments, the class Igene is a synthetic sequence selected for the ability to produce aprotein which protects a target cell from attack from more than oneclass of NK cells. In preferred embodiments the transgene includes agene, e.g., a chimeric or mutated HLA C gene, which confers protectionto more than one class of NK cells, e.g., an allele of HLA C havingserine at position 77 and lysine at position 80, see e.g., Biassoni,1995, J. Exp. Med. Vol. 1-82:605-609, hereby incorporated by reference.See also Moretta et al., 1996, Ann. Rev. Immunol. 14:619-648, herebyincorporated by reference, which together with the disclosure herein,provides guidance for altering critical residues in the HLA C genes:

In yet other preferred embodiments the transgenc swine cell, tissue ororgan, includes one or more, or all of, of a transgene which encodes anBLA A gene, a transgene which encodes an HLA B gene, a transgene whichencodes an HLA C gene, and a transgene which encodes an HLA G gene.

Swine, cells, tissues, organs and other components of the invention caninclude a transgene which encodes a graft-supporting protein, e.g., ahuman growth factor or cytokine receptor, e.g., a growth factor orcytokine receptor involved in the regulation of hematopoiesis. Examplesof growth factor or cytokine receptor include the receptors for G-CSF,SCF, GM-CSF, IL-3, IL-6, IL-11, IL-2, Epo, and uteroferrin.

In other preferred embodiments the transgene encodes a graft-supportingprotein, e.g., a human adhesion molecule, e.g., an adhesion moleculeinvolved in engraftment and/or maintenance of hematopoiesic cells.Examples of human adhesion molecules include VLA-4, c-kit, LFA-1, CD11a,Mac-1, CR3, CD11b, p150, p95, CD11c, CD49a, LPAM-1, CD49d, CD44, CD38,and CD34.

In yet other preferred embodiments the transgene encodes a recipient ordonor protein, e.g., a cytokine, which directly, or indirectly (e.g., bythe stimulation or inhibition of the level of activity of a secondcytokine), inhibits an immune response mounted by donor cells againstthe recipient, e.g., IL-10, IL-4, IL-2, or TGF-β.

In yet other preferred embodiments the transgene encodes a chimericmolecule, e.g., a chimeric lymphokine, e.g., PIXY123.

In yet other preferred embodiments the transgene encodes agraft-supporting protein, e.g., a recipient or donor cytokine, whichdirectly, or indirectly (e.g., by the stimulation or inhibition of thelevel of activity of a second cytokine), inhibits an immune responsemounted by recipient cells against donor tissue; e.g., IL-10, IL-4,IL-2, or TGF-β.

In yet other preferred embodiments the transgene inhibits the expressionof action of a gene product which is graft-antagonistic, e.g., bydecreasing the expression of the gene product. For example, thetransgene is a mutationally inactivated copy of a gene which encodes adonor graft antagonistic protein, e.g., the donor cells' B-7 receptor,CD27 receptor, or LFA-3 receptor, or a donor receptor for a hostcytokine, and which when inserted into the donor genome, e.g., byhomologous recombination, results in an endogenous gene which ismisexpressed or which is mutationally inactivated, by, e.g., theintroduction of a mutation, e.g., a deletion, into an endogenous genomiccopy of the gene which encodes the donor cells' B-7 receptor, CD27receptor, or LFA-3 receptor, or a donor receptor for a host cytokine.

The transgene can be one which encodes an anti-sense RNA which, directlyor indirectly, inhibits the expression or action of a recipient-derivedgraft-antagonistic protein, e.g., an antisense RNA which inhibits theexpression of a donor-encoded B-7 receptor, CD27 receptor, or LFA-3receptor, or a donor receptor for a host cytokine.

The transgene can be one which encodes a dominant negative mutation in agene product which is graft-antagonistic, e.g., a donor cell receptorfor a host cytokine or donor B-7 receptor, CD27 receptor, or LFA-3receptor.

In yet other preferred embodiments the transgene includes a nucleic acidencoding a human peptide, e.g., a hematopoietic peptide, operably linkedto: a promoter other than the one it naturally occurs with, a swinepromoter, e.g., a swine hematopoietic gene promoter, a viral promoter;or an inducible or developmentally regulated promoter.

EXAMPLES

Materials and Methods

Animals. In order to assure diversity of the MHC at the outset (since atleast two different MSLA homozygous herds were desired), the initialbreeds were chosen from different independently established herds ofminipigs. The initial boar, pig 1, was purchased from Vita VetLaboratories, Marion, Ind., and the initials sow, pig 2, was a “pigmeepig” purchased from the Hormel Institute, Austin, Minn. The animals werehoused indoors in 10-×14-foot pens on concrete floors and were fed onPurina complete sow chow. Pregnant sows were moved into separate boxstalls for farrowing.

Immunization. Initial typing antisera were obtained by full thicknessskin grafting between pigs 1 and 2, prior to breeding. The animals wereanesthetized with ketamine and halothane and a 3-inch square, fullthickness skin graft was transferred reciprocally between theirposterior thoraces. Serum samples were drawn prior to grafting and atweekly intervals thereafter.

Booster injections with approximately 108 live peripheral lymphocyteswere performed after cytotoxic titers had plateaued following skin graftrejection. Lymphocytes were obtained from approximately 50 ml ofheparinized donor blood by the Ficoll-Hypaque sedimentation method ofBoyum (described in Boyum A. (1968) Scand. J. Clin. Lab. Invest. 21:97).These were injected intramuscularly into the recipient animal and serumwas obtained at weekly intervals thereafter.

Small blood samples (up to 10 ml) were obtained by venipuncture of earveins. Larger samples were obtained by venipuncture of the anterior venacava with the animal in a supine position.

Technique for Skin Grating

Split-thickness skin grafts (STSG) measuring approximately 0.4 mm×6.0cm×4.0 cm were taken from the dorsal surface of the ear and placed on afull-thickness graft bed on the posterior thorax. Each minipig receivedan autograft and an allograft, held in place by a compression dressingof Vaseline gauze. Dressings were removed on the third day, and graftswere inspected daily until rejection was complete. Serum was obtainedfrom each minipig prior to grafting and at regular intervals thereafter;all sera from a skin allograft recipient were tested against donorlymphocytes in the two-stage cytotoxic assay.

Serology. Hemagglutination assays were performed in a crossmatchfashion, adding a few μ1 of washed packed red cells to a drop of freshplasma, incubating at 37° C. for 10 minutes, and scoring agglutinationfrom 1⁺ to 4⁺ under low power light microscopy.

Trypan blue cytotoxicity tests were performed in disposable U-bottomMicrotiter plates (Cooke Engineering Co., Alexandria, Va.) usinglymphoid cell suspensions obtained by Ficoll-Hypague sedimentation offresh heparinized and passage through loosely packed washed glass wool.A two-stage cytotoxic assay using rabbit complement was performed aspreviously (described in Sachs D. H. et al. (1971) J. Immunol. 107:481).

In vitro absorptions of antisera were performed by mixing appropriatenumber of lymphoid cells with antiserum in a 15-ml conical centrifugetube. The cells and serum were mixed and incubated at 4° C. for 0.5hour, with mixing at 15 minutes, and the tube was then centrifuged at900 g for 15 minutes to yield an absorbed antiserum.

Mixed lymphocyte cultures. Lymphocyte separations were prepared asabove, except that a sterile technique was used for the venipuncture andthroughout the preparation of the lymphocyte suspension. Tissue culturemedium consisted of RPMI 1640 with 100 units of penicillin per ml, 100μg of streptomycin per ml, and 5% fetal pig serum.

For one-way MLC reactions, one-half of each cell suspension was eitherirradiated with 2,000 R from a “Gammator M” cesium source (IsomedixInc., Parsippany, N.J.) or was incubated at 37° C. for 30 minutes with25 μg of mitomycin C (Nutritional Biochemical Co., Cleveland, Ohio) per5×10⁶ cells and was washed 5 times with medium. Cell suspensions wereadjusted to 5×10⁶ cells/ml and 0.1-ml aliquots of each cell suspensionwas mixed in the wells of V-bottom Microtiter plates (Cooke EngineeringCo.). Controls consisted of 0.1 ml of treated responder cells from thesame animal. All test combinations were run in triplicate. Plates wereincubated in 100% humidity at 37° C. with 5% CO2 in air in a Nationalincubator. Each well was pulsed with 1 μCi of tritiated thymidine(Amersham/Searle Corp., Arlington Heights, Ill.) for 4 hours on the 5thday, and cultures were then harvested with a MASH II (MicrobiologicalAssociates, Bethseda, Md.) harvester. Liquid scintillation counting wasperformed in Yorktown Hydromix solution. Results were expressed asratios of experimental cpm versus control cpm. The control for one-wayMLCs was the activity incorporated in autologous cultures describedabove. The control values for two-way MLCs were taken as the sum ofone-half of the incorporation of each of the control cultures for thetwo cells tested.

Example 1 Production of Homozygous Miniature Swine Leukocyte Antigen(MSLA) Herds

Serology. Natural anti-red cell antibodies were found to be present inseveral combinations of pigs that were tested. Unlike the situation inman, these red cell antibodies appeared to be cytotoxic to lymphocytesunder the conditions of the cytotoxic assay. Table 1 shows a typicalexample of the results obtained with preimmune serum in such acombination. TABLE 1 Lymphocytotoxicity of natural antibodies Absorptionwith Agglutination of Lymphocytotoxicity on target strain lymphocytesImmunization status target strain target strain Medium ComplementAntiserum dilution of serum donor red cells red cells control control 12 4 8 16 32 64 128 256 Pre − +2 <10 <10 70 71 55 22 <10 <10 <10 <10 <10immune + 0 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 Pre − +2 <10<10 >80 >80 >80 >80 >80 >80 74 22 <10 immune + 0<10 >10 >80 >80 >80 >80 >80 >80 51 13 <10

As indicated, when the hemagglutinatin was positive on red cells, a lowtiter of cytotoxic antibody was also seen in the preimmune serum.Absorption of such sera with five parts of packed washed red cells wasfound to remove both the hemagglutinating antibodies and the cytotoxicactivity. Cytotoxic titers of postimmune antisera (also indicated inTable 1) were not altered by a similar red cell absorption, indicatingthat the converse was not true, i.e., anti-MSLA antibodies are notabsorbed significantly by pig red cells.

Unlike many mammalian species for which normal rabbit serum frequentlycontains large amounts of natural cytotoxic antibodies, satisfactoryrabbit complement was readily obtained for miniature swine lymphocytes.Serum from each of four outbred New Zealand rabbits tested produced lessthan 10% background cytotoxicity at a 1:2 dilution of complement andprovided adequate complement to give complete cytotoxic lysis ofsensitized target cells to a dilution of greater than 1:8. This makes iteasy to work in relative complement excess for this species. Inaddition, miniature swine lymphocyte preparations obtained byFicoll-Hypaque separation of peripheral blood were found to be verysatisfactory target cells for cytotoxic assays. These cells remainedviable in the medium used for cytotoxic assays (Medium 199 containing0.1% gelatin) for at least 2 days at 4° C.

Course of cytotoxic antibody production. Animals that received skingrafts for immunization showed rejection by visual inspection between 8and 10 days. Cytotoxic antibodies appeared in the host serum by 2 weeksafter grafting and remained elevated for several weeks thereafter.Following a boost with peripheral lymphocytes, the titer of cytotoxicantibodies generally rose by several 2-fold dilutions and then againplateaued. A typical pattern of the course of cytotoxic antibodiesfollowing skin grafting and boosting in these miniature swine shown inFIG. 1. Recipient pig's serum was tested for cytotoxicity against donorlymphocytes each week after grafting, using the standardmicrocytotoxicity assay as described in Sachs, et al. (1976)Transplantation 22:559-567. Cytotoxic titers were taken as the lastdilution of sera producing more than 50% lysis of target cells.

Serological analysis of first generation offspring. In a wide variety ofmammalian species that have been studies so far, rejection of allograftsis accompanied by the appearance in the recipient's serum of antibodiesdetecting products of the MHC antigens of the donor. Assuming that thereis one MHC in miniature swine and that the initial animals chosen forbreeding differed in alleles at this locus, we can assign letters forthe genotypes at the MHC of each of the pigs arbitrarily as AB for pig 1and CD for pig 2. This represents the maximum heterogeneity that wouldbe possible for a single autosomal locus. The offspring of pig 1 and pig2 would thus be of 4 possible genotypes, as indicated schematically inFIG. 2.

If the immunization of pig 1 (AB) with tissue from pig 2 (CD) producedantibodies detecting both alleles at the MHC, we could expect theantibodies to consist of anti-C and anti-D components. Similarly, pig 2(CD) immunized with pig 1 (AB) should produce anti-A and anti-Bantibodies. It should thus be possible by absorption studies todetermine which of the offspring inherited each of the theoreticallypossible alleles, as shown schematically in FIG. 2 for one of the sera.Serum 308 (pig 1, anti-pig 2) and serum 309 (pig 2, anti-pig 1) weretested by such an absorption analysis on each of the offspring that theyproduced. The results with serum 308 tested on four of the offspring(assigned identification numbers 5, 6, 8, and 9) that were subsequentlychosen for further breeding are shown in Table 2. TABLE 2 Absorptionpattern of antiserum 308 (pig 1 anti-pig 2) in pigs of the firstgeneration Complement Lysis after absorption with Test Cells controlNone 5 6 8 9 5 <10 >80 47 57 >80 >80 6 <10 >80 16 <10 >80 >80 8<10 >80 >80 >80 60 47 9 <10 >80 >80 >80 52 56

Despite the incompleteness of the absorptions, it was clear from theseresults that the four offspring could be separated into two groups, eachhaving received one of the possible MSLA alleles from parent 2. Pigs 5and 6 were therefore arbitrarily assigned MSLA haplotype C and pigs 8and 9 were assigned the haplotype D.

However, when a similar absorption study was carried out with serum 309,all of the offspring of this mating were found capable of absorbingcytotoxic reactivity against all of their siblings, as indicated inTable 3. TABLE 3 Absorption pattern of antiserum 309 (pig 2 anti-pig 1)in pigs of the first generation Complement Lysis after absorption withTest cells control None 5 6 8 9 5 <10 >80 23 25 23 37 6 <10 >80 <10 <10<10 <10 8 <10 >80 <10 <10 17 16 9 <10 >80 11 <10 16 13

Repeated absorptions with increasing numbers of lymphocytes showed thesame pattern of mutual reactivity of all the siblings. It thereforeappeared that only one haplotype was transmitted from pig 1 to all ofthe offspring in this mating and only one haplotype designation, A,could be assigned to all of these offspring. It seems possible eitherthan pig 1 was in fact a homozygote at the MHC (i.e., AA) or that he wasa heterozygote (AB) but that only the A allele was transmitted to hisoffspring. The latter possibility could have been due to chance alone(P=0.06) or because of unknown selective pressures. As indicated below,all of the subsequent typing data obtained, confirmed the transmissionof only a single MSLA haplotype from pig 1.

Serological typing of subsequent generations. Only three possible MSLAgenotypes would be expected from the breeding of two identicalheterozygotes (i.e., AC×AC−AA+2AC+CC). In addition, antisera 308 and 309should be essentially monospecific with respect to their reactions withsuch offspring. Therefore, no absorptions of these sera were required inorder to determine which allele each had inherited. A typical example ofthe genotyping of one litter obtained from pigs 5 and 6 is indicated inTable 4. TABLE 4 Typing of the second generation Maximum % lysis by:Serum 308 Serum 309 Comple- (pig 1, anti- (pig 2, anti- Off- ment pig 2,anti- pig 1, DC Assign- Parents sping control AA-CD) anti-AA) ment Pig 5(AC) × 42(M) <10 >80 <10 CC Pig 6 (AC) 43(F) <10 <10 >80 AA 44(M)<10 >80 >80 AC 45(F) <10 >80 >80 AC 46(F) <10 >80 <10 CC 47(M)<10 >80 >80 AC 48(F) <10 >80 >80 AC 49(F) <10 >80 <10 CC 50(F)<10 >80 >80 AC Pig 8 (AD) × 23(M) <10 >80 <10 DD Pig 6 (AD) 24(M)<10 >80 <10 DD 25(F) <10 >80 <10 DD

Similar serological typing was carried out for all of the offspringobtained from these breedings. FIG. 3 presents a summary of the genotypeassignments of the miniature swine that have thus far been obtained fromthe original pigs 1 and 2. It is apparent from this figure that, by thethird generation, approximate breeders had been obtained to producethree herds of miniature swine, each homozygous for a different set ofMSLA genes.

MLC typing. In other mammalian species so far studied, including man,mouse, rat guinea pig, dog, and domestic swine, mixed lymphocyte culture(MLC) stimulation has correlated with serological typing for the MHC. Ittherefore seemed probable that if the MHC was indeed being typed forserologically, MLC reactions should corroborate the genotyping. MLCtyping of the earlier generations of these miniature swine was carriedout under suboptimal conditions, using 5% fetal calf serum rather thanfetal pig serum. Stimulation levels were low, but neverthelesssignificant and reproducible. After introduction of fetal pig serum intothe medium, stimulations rose from 2-fold to as high as 10- to 20-fold.In all cases there was concordance between MLC reactivity ad theexpected genotype at the MHC obtained by serological cytotoxicitytyping. Table 5 shows the MLC data obtained for the first generationoffspring of pigs 1 and 2. TABLE 5 MCL reactions of first generationoffspring Control cpm Stimulation ratio Cells cultured (½A + ½B)Experimental (experimental/control) 5(AC) + 6(AC) 2,757 ± 546  2,966 ±1,079 1.08 5(AC) + 8(AD) 5,795 ± 1,177 13,287 ± 1,013 2.29 5(AC) + 9(AD)6,116 ± 1,453 11,811 ± 3,465 1.93 6(AC) + 8(AD) 4,464 ± 931 10,151 ±1,434 2.28 6(AC) + 9(AD) 4,785 ± 1,207 11,216 ± 449 2.35 8(AD) + 9(AD)7,723 ± 1,838  7,401 ± 1,723 0.96

Despite the low stimulation values obtained, it is clear from these datathat the same assignment of genotypes as was made on the basis of thecytotoxicity typing can account for the patterns of MLC stimulationseen. Table 6 shows a more recent analysis of MLC reactions betweenseveral animals homozygous for CC by serological analysis of MSLAgenotypes (see FIG. 3). TABLE 6 MLC reactions of serological homozygotesCells cultured Control Experimental cpm Stimulation ratio 42(CC) +66(DD) 1,593 ± 319  23,372 ± 2,607 14.67 46(CC) + 66(DD) 2,677 ± 80910,090 ± 458  3.77 68(CC) + 66(DD) 1,025 ± 180  11,658 ± 1,379 11.3777(CC) + 66(DD) 1,151 ± 74  4,921 ± 353 4.28 42(CC) + 46(CC) 2,899 ± 5643,709 ± 654 1.28 42(CC) + 68(CC) 2,113 ± 231 2,055 ± 492 0.97 42(CC) +77(CC) 2,147 ± 287 1,610 ± 304 0.75 46(CC) + 68(CC) 1,891 ± 476 2,337 ±434 1.24 46(CC) + 77(CC) 1,922 ± 532 1,466 ± 159 0.76 68(CC) + 77(CC) 1,139 ± 198. 1,374 ± 229 1.20

Since the animals were taken from different sibships, this type ofanalysis provides a stringent test for the absence of other geneticfactors causing MLC reactivity not associated with the MHC. As can beseen from these data, no significant stimulation was obtained in two-wayMLC reactions between the MSLA homozygous identical animals. Also shownin the table are one-way control MLC reactions in which each of the CChomozygotes were found to be capable of mounting a significant responseto DD cells, indicating that the nonresponsiveness seen in the two-waycultures was not due to inability of any of the CC cells to proliferate.In addition, a variety of one-way MLC reactions were performed betweenhomozygous and heterozygous animals sharing one MSLA haplotype (e.g.,AA+ACx and AC+AAx). In such experiments significant stimulation ratioswere obtained only in the direction of AA+ACx. Thus the data areentirely consistent with the presence of a strong MLC stimulatory locus(or loci) within or closely linked to the serologically defined MSLAlocus.

Example 2 Production of Miniature Swine Homozygous at MHC (haplotype D)and 85% Homozygous at all other Genetic Loci

In this example, miniature swine homozygous for the MSLA haplotype Dwere first mated in a strictly non-brother-sister fashion for 20-25generations and then strictly brother-sister mated for 7 generations inorder to obtain a herd of animals homozygous at other genetic loci.Split-thickness skin grafts were used to quantitate the percentage ofinbreeding. By the seventh generation, animals approximately 85%homozygous at all other genetic loci were obtained.

Example 3 Cocultivation of Miniswine Peripheral Blood Mononuclear Cellswith Human 293 Cells and Porcine ST Iowa Cells

Objective

The purpose of this study was to cocultivate mini swine peripheral bloodmononuclear cells, (PBMCs) with the porcine endogenous retrovirus (PERU)susceptible human cell line 293 (ATCC CRL-1572) and with the porcinesusceptible cell line ST Iowa (ATCC CRL-1746).

Rationale

A safety consideration in xenotransplantation procedures is the possibletransmission of a zoonotic infectious agent from donor to recipient.Recent publications have described the detection of endogenousretrovirul sequences in pigs, a source of cells and organs inxenotransplantations. Additionally some continuous porcine cell lines,which contain similar retroviral sequences, produce PERV which isinfectious to other cell types, including human 293 cells. Consequently,it is prudent to assess the infectivity, if any, of, PERV relatedsequences present within the genomes of porcine cells which are to beused in xenotransplantation.

Experimental Design

A. Study Overview

Isolated pig peripheral blood mononuclear cells (PBMCs) were activatedfor 3 days with phytohemaggluttinin (PHA), phorbol myristate acetate(PMA), and IL-2. Subsequent to activation, PBMCs, both irradiated andnon-irradiated, were cocultivated with the cell lines 293 (a transformedhuman embryonic kidney cell line) and ST Iowa (a continuous porcinetestis cell line).

The cocultivation lasted for approximately 35 days. Cultures werepassaged when they were 90 to 100% confluent, (but not prior to day 6).On approximately days 6, 21, and 35, cell free supernatants wereharvested from all cultures. This timeline was subject to the conditionthat supernatants cannot be harvested until 2 days post passaging of theculture.

Harvested cell free supernatants from days 6, 21, and 35 were analyzedby RT-PCR for porcine endogenous retrovirus (PERV). Enzymatic RT assayswere performed on day 35 cell free supernatants.

On approximately day 21, approximately five fold concentrated cell freesupernatants from some cultures were inoculated onto polybrened,subconfluent 293 monococultures. Blind passaged cultures included PBMCmonocultures, PBMC and irradiated PBMC/293 cocultures, PBMC andirradiated PBMC with ST Iowa, the 293, ST Iowa and PK-15 (ATCC CCL-33)monocultures and the irradiated PK-15/293 coculture. These blind passagecultures were maintained for 21 days and passaged when necessary. At day21, RT-PCR for PERV was performed on cell free supernatants and DNA PCRfor PERV was performed on cells.

Cells were harvested from each of the surviving cultures in the study onthe last day of the study, and DNA was extracted from cells. DNAs wereamplified with pig specific multicopy gene primers to determine ifporcine cells were present in the cocultivations.

B. Specific Procedures

1. Initiation of Monocultures and Cocultures

a. Activation of PBMCs

PBMCs from mini swine blood were isolated by Ficoll density gradientseparation. Washed buffy coat cells were counted and aliquoted into T25flasks. Five ml of 1×106 PBMCs/ml were placed in each flask. Mediumconsisted of RPMI 1640, 15% irradiated FBS, L-glutamine, antibiotics,IL-2, PHA and PMA. Cells were incubated at 7-10% CO2, at 37° C. PBMCsremained in this medium for 3 days.

b. Seeding Procedures

After 3 days aliquots of cells were tested for thyrnidine uptake. Theactivated cells were plated into P 100 tissue culture dishes and allowedto sit for 4-6 hours to remove adherent cells. Aliquots of approximately5×10⁶ viable non-adherent PBMCs were pelleted by centrifuging at asetting of 800 rpm for 8 minutes at room temperature. Each aliquot ofpelleted cells was resuspended in 5 ml of medium consisting of heatinactivated DMEM with 10% FBS, L-glutamine, antibiotics, and IL-2.

Aliquots of 5 ml of cell suspension, each containing 5×10⁶ PBMCs, wereseeded as follows:

-   (1) One aliquot was placed into each of four T25 flasks. Two flasks    were X-irradiated for the appropriate period of time (approximately    2000 rads). Two flasks remained unirradiated.-   (2i) 1.0×10⁶ 293 cells were placed into each of 2 sterile 15 ml    centrifuge tubes. The cells were pelleted by centrifugation and the    supernatants discarded. Each of two pellets of 293 cells were    resuspended in an aliquot of 5 ml of 5×10⁶ PBMCs. After gentle    mixing, one cocultivation was placed into each of two T25 flasks.-   (2ii) 1.0×10⁶ 293 cells were placed into each of 2 sterile 15 ml    centrifuge tubes. The cells were pelleted by centrifugation and the    supernatants discarded. Each of two pellets of 293 cells were    resuspended in an aliquot of 5 ml of 5×10⁶ irradiated PBMCs    (approximately 2000 rads).

After gentle mixing, one cocultivation was placed into each of two T25flasks.

-   (2iii) 0.5×10⁶ ST Iowa cells were placed into each of 2 sterile 15    ml centrifuge tubes. The cells were pelleted by centrifugation and    the supernatants discarded. Each of two pellets of ST Iowa cells was    resuspended in an aliquot of 5 ml of 5×10⁶ PBMCs. After gentle    mixing, one cocultivation was placed into each of two T25 flasks.-   (2iv) 0.5×10⁶ ST Iowa cells was placed into each of 2 sterile 15 ml    centrifuge tubes. The cells were pelleted by centrifugation and the    supernatants discarded. Each of two pellets of ST Iowa cells was    resuspended in an aliquot of 5 ml of 5×10⁶ irradiated PBMCs    (approximately 2000 rads). After gentle mixing, one cocultivation    was placed into each of two T25 flasks.    c. The Positive Control Cultures were as Follows:

Duplicate flasks of PK-15 cells seeded the preceding day at 1×10⁶ cellsper flask were X-irradiated (approximately 2000-10,000 rads). Followingirradiation, 1.0×10⁶ 293 cells per flask were overlaid on the PK-15cells.

Duplicate flasks of unirradiated PK-15 cells seeded at 0.5×10⁶cells/flask.

d. The Negative Control Cultures were as Follows:

Duplicate cultures of unirradiated 293 cells seeded at 1.0×10⁶cells/flask. Duplicate cultures of unirradiated ST Iowa cells seeded at0.5×10⁶ cells/flask.

All cultures were maintained without passaging for 6 days postinitiation of cocultures. Six days post initiation of cocultures, allcultures that were 90% or more confluent were passaged using a splitratio appropriate to the cell type.

PBMC cultures were considered 100% confluent when the cell densityreached 2×10⁶ cells/ml. Cultures which are less than 90% confluent wererefed as necessary until they were 90-100% confluent, at which time theywere passaged. 293 and ST Iowa monocultures, 293/PBMC cocultures, and STIowa/PBMC cocultures were split a total of 5 times prior to day 34 postirradiation.

After day 6 cultures were maintained in complete 293 medium (DMEM, 10%heat inactivated FBS, 2-4 mM L-glutamine and 1% antibiotics) at 36OC and10% CO2 for the duration of the study.

e. 1.0×10⁶ 293 cells were inoculated with approximately 5 foldconcentrated cell free supernatants from day 21 harvests. These cultureswere maintained and split as necessary for 21 days. Cellfree-supernatants and cellular DNA were harvested from these blindpassage cultures on day 21. See Table 7. TABLE 7 Number of Cells inTreatment Irradiation Flask (×10⁻⁶) Number of Flasks ST Iowa No 0.5 2293 No 1.0 2 PBMC No 5 2 PBMC Yes 5 2 PBMC No 5 2 ST Iowa No 0.5 PBMC No5 2 ST Iowa No 0.5 PBMC Yes 5 2 293 No 1.0 PBMC No 5 2 293 No 1.0 PK-15Yes 1 2 293 No 1 PK-15 NO 0.5 22. Cell Harvesting and DNA Extraction

Cell viabilities were performed on all monocultures and cocultures onthe last day of the cocultivation and the last day of the blind passage.Cultures were then centrifuged, or trypsinized and centrifuged, topellet cells. To the cell pellets from each of the nine treatments, a 1×lysis buffer was added. Samples were incubated at 56° C.±2° C. for 30minutes and 1-2 hours at 37° C. Lysed samples were phenol/chloroformextracted and precipitated in NaCl and ethanol at −16° C. or below or at−70° C. to −80° C. Samples were centrifuged at a setting of 4° C. Thepellets were rinsed with 70-75% ethanol, and centrifuged again at asetting of 4° C. The pellets were air dried.

DNA pellets were dissolved in sterile Tris EDTA (TE) so that DNAequivalent to approximately 1×10⁵ cells (approximately 800 ng of DNA)existed in each 10 (1 sample. Samples were frozen at −70° C. or belowuntil use.

3. Harvesting of Cell Free Supernatants

At all time points, cell free culture supernatants were harvested in thefollowing manner: supernatant was harvested, centrifuged at a setting of800 rpm for 10-15 minutes, transferred to new tubes and then centrifugedat 2000×g for 15 minutes, filtered through a 0.45 (m cellulose acetatefilter, aliquoted and frozen at −70° C. until assayed, if not assayedimmediately.

Detection of Porcine Endogenous Retroviruses in Cell CultureSupernatants using Reverse Transcription and Polymerase Chain Reaction

Objective

The purpose of this study was to detect RNA sequences specific to theprotease region of porcine endogenous retroviruses (PERV) that may existin pig cells, tissues or organs (Test article).

Rationale

A safety consideration in xenotransplantation procedures is the possibletransmission of a zoonotic infectious agent from donor to recipient.Recent publications have described the detection of endogenousretroviral sequences in pigs, a source of cells and organs inxenotransplantations. Additionally some continuous porcine cell lines,which contain similar retroviral sequences, produce PERV which isinfectious to other cell types, including human 293 cells.

A sensitive and rapid method for detection of viral RNA and/or RNAtranscripts from. proviral retroviruses is RT-PCR. This protocoldescribes the methods used when testing cell culture fluids for PERVspecific RNA sequences.

Experimental Design

A. Sample Preparation

An aliquot of each test article was spiked with positive control RNAprior to extraction in order to show recovery of PERV RNA. In the caseof samples from co-cultivation studies, only supernatants from“indicator” cultures test were spiked.

RNA preparation was performed by pipetting supernatants vigorously inthe presence of guanidine isothiocyanate and phenol. Chloroform wasadded and the sample mixed. Samples were then centrifuged. The aqueousphase was transferred to a new tube and isopropanol added. Samples wereincubated for approximately 10 minutes at room temperature and thencentrifuged. RNA pellets were washed once with 75% ethanol andcentrifuged again. RNA pellets were partially air dried before beingresuspended.

The pellets were dissolved in DNAse free water or buffer. A DNAsereaction buffer and RNAse free DNAse were added to the sample. Sampleswere incubated for the appropriate period of time at 37° C.±2° C.Subsequent to incubation, the samples were briefly heated to 95° C.±2°C. The samples were then be assayed immediately or frozen at −60° C. orbelow until use.

B. Preparation of Polymerase Chain Reaction Solution

Polymerase chain reaction (PCR—mixtures (master mix) were prepared inthe PCR master mix room. Only preparations of master mix occur in thisroom. Master mix was comprised of sterile molecular biology grade water,reaction buffer, the nucleotides dCTP, dATP, dGTP and dUTP and theenzyme Tfl polymerase (Promega, Madison, Wis.). Uracil DNA Glycosylase(UDG, New England Biolabs, Beverly, Mass.) may also be used in thereaction buffer to prevent carryover. Two separate master mixes wereprepared. Each contained reagents as above. Reverse trancriptase wasadded to one set of master mixes. Water in its place was added to thesecond set of master mix. Both of these mixes contained primers specificto the protease portion of a porcine endogenous retrovirus (PERV)(Patience et al., 1997 Nature Medicine Vol 3 Number 3).

C. Addition of Test and Control Articles to PCR Reaction Mixtures

Master mixes were aliquoted into numbered tubes. Tubes were thentransported to the appropriate rooms for addition of test and controlarticles. Five to ten (1 of test or control article were added to eachtube. Tubes were cross referenced by number and sample identification inthe documentation. Test and control samples may be heated to 70° C.±2°C. for about 10 minutes to denature the RNA prior to adding to thereaction mixtures.

Reactions which contained protease-specific PERV primers and reversetranscriptase included the following test and control articles:

-   Master Mix only-no template RNA-   Negative control RNA from tobacco mosaic virus or other RNA virus,    run in duplicate. 5-10 (1 of extracted and DNAse treated test    article RNA(s).-   Test article RNA or cocultivation indicator cell derived RNA spiked    with at least the 10-2 dilution of positive control PERV RNA.-   Extracted, DNAse treated RNA from positive control PERV stock in    dilutions of at least 10⁻², 10⁻³, 10⁻⁴.

Reactions which contained protease specific PERV primers without reversetranscriptase included one replicate of the following test and controlarticles:

-   Test article RNA(s)-   Spiked test article RNA spiked or cocultivation indicator cell    derived RNA. Positive control RNA from the lowest dilution of PERV    used in the assay    D. Amplification, Gel Electrophoresis and Southern Transfer

If UDG was added to the reaction mixtures the tubes were placed in athermal cycler and incubated at 22° C.±2° C. to allow the UDG to act,then incubated at 95° C.±2° C. for 2 minutes prior to cDNA synthesis.cDNA synthesis occurred at 48° C. for 40-60 minutes. Reaction mixtureswere then amplified through 35 to 40 cycles of denaturation, annealingand extension temperatures. Gel loading dye was added to the samples andthe samples, along with DNA size standards, were electrophoresed onagarose gels. Ethidium bromide stained gels were observed andphotographed on an UV transilluminator. Subsequent to gel staining,Southern transfer procedures were used to transfer samples amplifiedwith the protease specific PERU primers to a nylon membrane. Themembranes were UV crosslinked and, if not used immediately, wrapped andstored at 4° C.±2° C. until use.

E. Detection

The membranes were hybridized to a fluorescein-11-tagged oligonucleotideDNA probe. After washing and blocking, the membrane was incubated in asolution containing an antifluorescein-horseradish peroxidase conjugate.Signal generation and detection occurs with the addition ofcherniluminescent detection solutions and subsequent exposure of themembrane to X-ray film.

Test System

cDNAs were prepared from test article derived RNA. These cDNA were thenanalyzed by use of the polymerase chain reaction (PCR), followed by gelelectrophoresis, Southern transfer, hybridization with anoligonucleotide probe and subsequent detection by chemiluminescence.

Control Articles

Positive Control Article

-   1. Identification: PERV RNA produced from PK-15 (ATCC # CCL-33)    culture fluids-   2. Source: GTC Washington Laboratories-   3. Storage Conditions: −60° C. or below    Negative Control-Article-   1. Identification: RNA from Tobacco Mosaic Virus-   2. Source: Boehringer Mannheim-   3. Storage Conditions: −60° C. or below    Assay Acceptance Criteria    The assay is considered valid if the following results are obtained.    A. For Reaction Mixtures Containing Reverse Transcriptase and    Amplified with PERV Primers:-   1. No signal is observed in either the no-template control or the    negative control.-   2. A signal is observed in at least the 10-2 dilution of the PK-15    supernatant derived PERV RNA.-   3. A signal is observed in the test article or indicator cell    derived RNA spiked with the 10-2 dilution of PERV positive control    RNA.    B. For Reaction Mixtures which do not Contain Reverse Transcriptase    Amplified with PERV Primers:-   1. No signal is observed in the negative control.-   2. No signal is observed in the test article RNA(s).-   3. No signal is observed in the positive control.    Detection of Porcine Endogenous Retroviral Reverse Transcriptase    Activity    Objective

The objective of this assay is to determine whether retroviruses arepresent in the test article by analysis for retroviral reversetranscriptase activity.

Experimental Design

Reverse transcriptase was determined using duplicate reactions. Theincorporation of tritiated thymidine triphosphate into newly synthesizedDNA was measured using a synthetic template, poly (rA)-oligo (dT).Porcine endogenous retrovirus virus (PERV) and monkey retrovirus (SMRV,ATCC VR-843) (type D) were included as positive controls. Test articlesamples were diluted with stabilization buffer and/or medium in order todetermine if an inhibitor of reverse transcriptase activity is present.For the same purpose, test article samples were diluted with PERV.Reactions will contain Mn++ (for PERV) and Mg++ (for SMRV).

Assay Samples were as Follows:

-   A. Undiluted test article-   B. Test article diluted two-fold with stabilization buffer or medium-   C. Test article diluted two-fold with PERV Mn++-dependent positive    control)-   D. PERV diluted two-fold with stabilization buffer or medium-   E. SMRV (Mg++-dependent positive control)-   F. Stabilization buffer and/or medium (negative control)    Rationale

Retroviruses possess the enzyme RNA-dependent DNA polymerase (reversetranscriptase) which is capable of catalyzing the synthesis of DNA usingretroviral RNA as a template. The endpoint the assay utilizes is thequantitation of incorporated tritiated thymidine triphosphate into newlysynthesized DNA.

Protocol

The assays were performed as described in Phan-Thanh et al. PorcineRetrovirus Reverse Transcriptase Optimal Conditions for itsDetermination. Develop. Biol. Standard 72, 111-116, 1990.

Results

Data presented here are from the day 35 reverse transcriptase (RT)analysis and the day 42 RTPCR on cell free supernatants.

Monocultures and cocultivations were cultured for 35 days. At day 21cell free supernatants were inoculated onto polybrened, subconfluent 293monocultures (blind passage). Blind passage cultures were maintained for21 days. Cells were harvested at day 35 of the cocultivation and day 21of the blind passage. Day 21 of the blind passage is referred to as day42 in this study. Cell free supernatants were harvested at days 7 and35. Irradiated and unirradiated PBMC monocultures were harvested priorto day 21, consequently these two treatments were not blind passaged andno day 35 samples were available for these two treatments.

RNAs extracted from filtered and clarified day 42 cell supernatants weretranscribed into cDNAs and then amplified with primers specific forporcine endogenous retrovirus (PERV) sequences. Following RT-PCR,reaction mixtures were transferred to a nylon membrane and hybridizedwith a PERV specific oligonucleotide. FIG. 4 shows the results of RT-PCRanalysis of cell free supernatants derived from 293 cultures from day 21of the blind passage. No evidence of PERV specific RNA was seen insupernatants from 293 cultures inoculated with supernatants derived fromST Iowa cells, 293 cells or irradiated and non-irradiated PBMCscocultivated with 293 cells. PERV specific RNA was observed insupernatants from 293 cultures inoculated with supernatants derived fromirradiated PBMC/ST Iowa cocultivations and non-irradiated PBMC/ST Iowacocultivations. PERV specific RNA was also observed in PK-15 cells aloneand 293 cells cocultivated with PK-15 cells that had been irradiated attwo different doses of radiation. A positive signal was also observed inone of two replicates of 293 cells inoculated with supernatants fromPERV infected 293 cells. Positive signals were observed in 293 cellsinoculated with day 35 293 culture supernatants spiked with 2700 virusparticles. Positive controls equivalent to 2700 virusparticles(vp)/reaction and 270 vp/reaction and 27 vp/reaction resultedin positive signals.

Table 8 shows the results of enzymatic reverse transcriptase (RT)analysis of day 35 cell free supernatants. No evidence of RT activitywas seen in supernatants derived from indicator 293 cells alone or with293 cells cocultivated with PBMCs or irradiated PBMCs. TABLE 8 ReverseTranscriptase Activity¹ Non-irradiated PBMC293    560° Diluted 2-foldwith buffer   515 Diluted 2-fold with PERV 47,864 Irradiated PBMC293  514 Non-irradiated PBMCST Iowa_(—) 20,659 Diluted 2-fold with buffer11,139 Diluted 2-fold with PERV 68,221 Irradiated PBMGST Iowa 20,447 293  327 ST Iowa   1258^(a) Irradiated PK-15/293 33,995 Diluted 2-fold withbuffer   7322 Diluted 2-fold with PERV 56,630 Irradiated PK-15/29333,541 PK-15   4713 293 inoculated with PERV   493 PERV diluted 2-foldwith buffer 28,239 Stabilization buffer    573^(b)¹Mn++ dependent reverse transcriptase activity. Mean of duplicatesamples from one culture.Mean deviations are 10% or less except as follows: ^(a)31%;^(b)18%;^(c)16%.

When PBMCs were cocultivated with ST Iowa cells, levels or incorporationof 3H-TTP were 16 times greater than with ST Iowa cells alone. Whenirradiated PK-15 cells were cocultivated with 293 cells, the reversetranscriptase activity was at least seven times greater than withnon-irradiated PK-15 cells alone, indicating that either 293 cell wereinfected and PERV was amplified, or that the PK-15 cells had a greatercapacity for infection as a result of experimental conditions such asirradiation. No inhibition of PERV positive control was observed whencocultivated cells were diluted with PERV.

These results are in contrast to the results of Wilson et al. (1998, J.Virology vol 72, no. 4: 3082-3087) which reported that mitogenicactivation of PBMC from the National Institutes of Health (NIH) minipigand the Yucatan pig resulted in the activation and release of aninfectious type C retrovirus. Coculture of activated porcine PBMC withpig and human cell lines using the NIH minipig or the Yucatan pigresulted in the transfer and expression of PERV-specific sequences andthe establishment of a productive infection (Wilson et al. 1998, J.Virology vol 72, no. 4: 3082-3087).

Example 4 Microsatellite Analysis of Inbred Miniature Swine

Recently, mapping of microsatellite polymorphisms in mice and domesticlivestock animals has generated genetic maps which can be used formarker assisted selection of breeding pairs. In mouse, this hasfacilitated rapid construction of congenic inbred strains. In livestock,this has been employed to speed the process of generating strains withcommercially important traits. Additionally, microsatellite markers canbe used to rapidly detect recombination events (e.g. with the MHCcomplex) and to distinguish animals at an early stage (e.g., inembryo/fetal populations).

Short Tandem Repeats (STR's) are efficient tools for mapping specifictraits or to follow the flow of genetic material in a population. Thetechnology is based on the presence of short tracks of di, tri, tetra orpenta nucleotide repeats which are common in the genomes of eukaryoticorganisms. These short tracks (5 to 10 repeating units) are faithfullytransmitted thought sexual reproduction, but are often highlypolymorphic within a population.

High-throughput analysis of STR loci can be performed by firstamplifying the loci using flanking PCR primers. The size of loci (andhence the characteristic number of repeats) can be identified followingelectrophoretic separation on an ABI 377 sequence detector, providedfluorescent primers are used to tag the amplified products.

A pilot study of swine microsatellite markers in a highly inbred pair ofminiature swine has been performed. Genomic DNA was isolated fromminiature swine #s 13220 and 13222 (inbred at the SLA haplotype). Using225 pairs of primers (obtained from Professor Max Rothschild, Iowa StateUniversity, Ames Iowa) PCR products were generated. The PCR productswere sized and analyzed by Lark Technologies Inc. (Houston, Tex.). Theresults of the genotype analysis are presented in Table 9. TABLE 9Genotype Analysis Miniature Miniature swine swine #13220 #13222 Numberof different primer pairs used 225 225 in the PCR assay Number, ofdifferent primer pairs that 196 197 gave rise to PCR products Number (%)of different primer pairs 126 (64%) 147 (74%) that resulted in PCRproducts of the same length and as such, were considered to correspondto monomorphic alleles Number (%) of different primer pairs  70  50 thatresulted in PCR products of different lengths and as such, wereconsidered to correspond to dimorphic alleles Number (%) of differentprimer pairs  41***  41 that resulted in PCR products of differentlengths and as such, were considered to correspond to dimorphic allelespresent in both animals Number (%) of different primer pairs  29*  9**that resulted in PCR products of different lengths and as such, wereconsidered to correspond to dimorphic alleles different in the twoanimals*This number contains 13 loci with alleles differing by 2-3 bp**This number contains 2 loci with alleles differing by 2-3 bp***This number contains 16 loci with alleles differing by 2-3 bp.

As analysis of alleles that differ by 2-3 bp needs to be interpretedwith some caution further analysis should be performed to ascertainwhether the alleles are monomorphic or dimorphic. These resultstherefore indicate the minimum extent of inbreeding. Using theseresults, however, it can be determined that the coefficiency ofinbreeding for miniature swine #s 13220 and 13222 are 0.64 and 0.74,respectively.

These analyses provide a rationale for the inbreeding program in theselection of animals to maximize the extent of inbreeding.

Other Embodiments

The methods of the invention are particularly useful for replacing atissue or organ afflicted with a neoplastic disorder, particularly adisorder which is resistant to normal modes of therapy, e.g.,chemotherapy or radiation therapy. In preferred embodiments: the graftincludes tissue from the digestive tract or gut, e.g., tissue from thestomach, or bowel tissue, e.g., small intestine, large intestine, orcolon; the graft replaces a portion of the recipient's digestive systeme.g., all or part of any of the digestive tract or gut, e.g., thestomach, bowel, e.g., small intestine, large intestine, or colon.

Methods of the invention minimize or eliminate the need for preparativeWB irradiation. However, when irradiation is administered, it ispossible to induce mixed chimerism with less radiation toxicity byfractionating the radiation dose, i.e., by delivering the radiation intwo or more exposures or sessions. Accordingly, in any method of theinvention calling for the irradiation of a recipient, e.g., a primate,e.g., a human, recipient, of a xenograft, the radiation can either bedelivered in a single exposure, or more preferably, can be fractionatedinto two or more exposures or sessions. The sum of the fractionateddosages is preferably equal, e.g., in rads or Gy, to the radiationdosage which can result in mixed chimerism when given in a singleexposure. The fractions are preferably approximately equal in dosage.Hyperfractionation of the radiation dose can also be used in methods ofthe invention. The fractions can be delivered on the same day, or can beseparated by intervals of one, two, three, four, five, or more days.Whole body irradiation, thymic irradiation, or both, can befractionated.

Thymic irradiation can also be fractionated. For example, a single doseof 700 rads can be replaced with, e.g., two fractions of 350 rads, orseven fractions of 100 rads.

Methods of the invention can include recipient splenectomy.

As is discussed herein, hemoperfusion, e.g., hemoperfusion with a donororgan, can be used to deplete the host of natural antibodies. Othermethods for depleting or otherwise inactivating natural antibodies canbe used with any of the methods described herein. For example, drugswhich deplete or inactivate natural antibodies, e.g., deoxyspergualin(DSG) (Bristol), or anti IgM antibodies, can be administered to therecipient of an allograft or a xenograft. One or more of, DSG (orsimilar drugs), anti-IgM antibodies, and hemoperfusion, can be used todeplete or otherwise inactivate recipient natural antibodies in methodsof the invention. DSG at a concentration of 6 mg/kg/day, i.v., has beenfound useful in suppressing natural antibody function in pig tocynomolgus kidney transplants.

In any of the methods described herein, particularly primate or clinicalmethods, it is preferable to form mixed chimerism as opposed to entirelyreplacing the recipient's stem cells with donor cells.

Any of the methods referred to herein can include the administration ofagents, e.g., 15-deoxyspergualin, mycophenolate mofetil, brequinarsodium, or similar agents, which inhibit the production, levels, oractivity of antibodies in the recipient. One or more of these agents canbe administered: prior to the implantation of donor tissue, e.g., one,two, or three days, or one, two, or three weeks before implantation ofdonor tissue; at the time of implantation of donor tissue; or afterimplantation of donor tissue, e.g., one, two, or three days, or one, twoor three weeks after, implantation of a graft.

Preferred embodiments include administration of 15-deoxyspergualin (6mg/kg/day) for about two weeks beginning on the day of graftimplantation.

Some of the methods referred to herein include the administration ofhematopoietic stem cells to a recipient. The inventors have found thatadministration of one or more cytokines, preferably a cytokine from thespecies from which the stem cells are derived, can promote engraftment,mixed chimerism, and tolerance, or otherwise prolong acceptance of agraft. The use of such cytokines can reduce or eliminate the need forwhole body irradiation. Thus, the invention also includes methods in therecipient is administered one or more cytokine, e.g., a donor-speciescytokine.

Although not wishing to be bound by theory, the inventors believe thatthe cytokines, particularly donor species cytokines, promote theengraftment and/or function of donor stem cells or their progeny cells.Accordingly, any method referred to herein which includes theadministration of hematopoietic stem cells can further include theadministration of a cytokine, e.g., SCF, IL-3, or GM-CSF. In preferredembodiments the cytokine one which is species specific in itsinteraction with target cells.

Administration of a cytokine can begin prior to, at, or after theimplantation of a graft or the implantation of stem cells.

The method can further include the step of administering a fast orsubsequent dose of a cytokine to the recipient: when the recipientbegins to show signs of rejection, e.g., as evidenced by a decline infunction of the grafted organ, by a change in the host donor specificantibody response, or by a change in the host lymphocyte response todonor antigen; when the level of chimerism decreases; when the level ofchimerism falls below a predetermined value; when the level of chimerismreaches or falls below a level where staining with a monoclonal antibodyspecific for a donor PBMC antigen is equal to or fails below stainingwith an isotype control which does not bind to PBMC's, e.g. when thedonor specific monoclonal stains less than 1-2% of the cells; orgenerally, as is needed to maintain tolerance or otherwise prolong theacceptance of a graft. Thus, method of the invention can be modified toinclude a further step of determining if a subject is in need ofcytokine therapy and if so, administering a cytokine.

The period over which the cytokine(s) is administered (or the periodover which clinically effective levels are maintained in the subject)can be long term, e.g., for six months of more or a year or more, orshort term, e.g., for a year or less, more preferably six months orless, more preferably one month or less, and more preferably two weeksor less. The period will generally be at least about one week andpreferably at least about two weeks in duration.

In preferred embodiments the recipient is a primate, e.g., a human, andthe donor is from a different species, e.g., the donor is a pig and: pigSCF is administered; pig IL-3 is administered; a combination of pig SCFand pig IL-3 is administered; a pig specific hematopoiesis enhancingfactor, e.g., pig GM-SCF, is administered, e.g., after the implantationof stem cells, e.g., about a month after the implantation of stem cells.

A particularly preferred embodiment combines a short course, e.g., abouta month, of cyclosporine or a similar agent, a short course, e.g., abouttwo weeks, of 15-deoxyspergualin or a similar agent, and a short course,e.g., about two weeks, of donor specific cytokines, e.g., SCF and IL-3.In Cynomolgus monkeys receiving pig grafts and pig stem cells, treatmentwhich included the combination of cyclosporine (15 mg/kg/day for 28days), 15-deoxyspergualin (6 mg/kg/day for two weeks), and recombinantpig cytokines (SCF and IL-3, each at 10 mg/kg/day, i.v., for two weeks)was found to be useful. Administration began at the time of graftimplant. (The monkeys were also given a preparative regime consisting of3×100cGy whole body irradiation on day-6, and -5 and hemoperfusion witha pig liver just prior to stem cell administration.)

An anti-CD2 antibody, preferably a monoclonal, e.g., BTI-322, or amonoclonal directed at a similar or overlapping epitope, can be used inaddition to or in place of any anti-T cell antibodies (e.g., ATG) in anymethod referred to herein.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1.-23. (canceled)
 24. A method of preparing a transgenic swine,comprising: providing a swine which is homozygous at swine leukocyteantigens (SLA) A, B, C, DR, and DQ, and in which all other genetic lociare at least 60% homozygous; and introducing a transgene into saidswine, thereby preparing a transgenic swine.
 25. The method of claim 24,wherein said transgene encodes a human growth factor receptor.
 26. Themethod of claim 24, wherein said transgene encodes a cytokine receptor.27. A method of inducing tolerance in a recipient mammal of a firstspecies to a graft from a donor mammal of a second species, comprising:providing a donor mammal which is from a herd which is homozygous for amajor histocompatibility complex haplotype and at least 60% homozygousat all other genetic loci; introducing into the recipient mammal,tolerance inducing tissue; providing a graft from the donor mammal orfrom a second donor mammal from the herd; and introducing the graft intothe recipient, thereby inducing tolerance in a recipient mammal of afirst species to a graft from a mammal of the second species.
 28. Themethod of claim 27, comprising: providing a donor mammal which is from aherd which is homozygous for a major histocompatibility complexhaplotype and at least 60% homozygous at all other genetic loci; plintroducing into the recipient mammal, hematopoietic stem cells fromsaid donor mammal; providing a graft from said donor mammal or from saidherd; and introducing said graft into said recipient, thereby inducingtolerance in a recipient mammal of a first species to a graft from adonor mammal of a second species.
 29. The method of claim 27, whereinsaid recipient is a primate and said donor is a swine.
 30. The method ofclaim 27, wherein said recipient is a primate and said donor is aminiature swine.
 31. The method of claim 27, wherein said recipient is ahuman and said donor is a swine.
 32. The method of claim 27, whereinsaid recipient is a human and said donor is a miniature swine.
 33. Themethod of claim 29, wherein said swine is homozygous at swine leukocyteantigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of allother genetic loci are homozygous.
 34. The method of claim 27,comprising: providing a donor mammal which is from a herd which ishomozygous for a major histocompatibility complex haplotype and at least60% homozygous at all other genetic loci; introducing into the recipientmammal, thymic tissue from said donor mammal; providing a graft fromsaid donor mammal, or from said herd; and introducing said graft intosaid recipient, thereby inducing tolerance in a recipient mammal of afirst species to a graft from a donor mammal of the second species. 35.The method of claim 27, comprising: inserting a nucleic acid encoding anMHC antigen into a hematopoietic stem cell of the recipient, wherein thenucleic acid encodes an MHL antigen of a swine from a herd which ishomozygous for a major histocompatibility complex haplotype and at least60% homozygous at all other genetic loci; allowing the MHC antigenencoding nucleic acid to be expressed in the recipient; and preferably,implanting graft in the recipient, wherein the graft is from an animalfrom the herd.